Polymerase enzyme substrates with protein shield

ABSTRACT

Compositions and methods are provided for nucleotide analogs comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Nucleotide analogs of the invention have a protein shield between the dye moieties and nucleotide moieties of the analog. The protein prevents the direct interaction of the dye moiety with the enzyme carrying out nucleotide synthesis preventing photodamage to the enzyme. The nucleotide analogs of the invention can have multiple dyes and multiple nucleotide moieties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional U.S. PatentApplication No. 61/599,149, filed Feb. 15, 2012, the full disclosure ofwhich is incorporated herein by reference in its entireties for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED BY U.S.P.T.O. eFS-WEB

The instant application contains a Sequence Listing which is beingsubmitted in computer readable form via the United States Patent andTrademark Office eFS-WEB system, and is hereby incorporated by referencein its entirety for all purposes. The txt file submitted on Aug. 5, 2013contains only 6 KB file (01014601-2013-08-05AmendedSequenceListing.txt).

BACKGROUND OF THE INVENTION

The ability to read the genetic code has opened countless opportunitiesto benefit humankind. Whether it involves the improvement of food cropsand livestock used for food, the identification of the causes ofdisease, the generation of targeted therapeutic methods andcompositions, or simply the better understanding of what makes us who weare, a fundamental understanding of the blueprints of life is anintegral and necessary component.

A variety of techniques and processes have been developed to obtiingenetic information, including broad genetic profiling or identifyingpatterns of discrete markers in genetic codes and nucleotide levelsequencing of entire genomes. With respect to determination of geneticsequences, while techniques have been developed to read, at thenucleotide level, a genetic sequence, such methods can be time-consumingand extremely costly.

Approaches have been developed to sequence genetic material withimproved speed and reduced costs. Many of these methods rely upon theidentification of nucleotides being incorporated by a polymerizationenzyme during a template sequence-dependent nucleic acid synthesisreaction. In particular, by identifying nucleotides incorporated againsta complementary template nucleic acid strand, one can identify thesequence of nucleotides in the template strand. A variety of suchmethods have been previously described. These methods include iterativeprocesses where individual nucleotides are added one at a time, washedto remove free, unincorporated nucleotides, identified, and washed againto remove any terminator groups and labeling components before anadditional nucleotide is added. Still other methods employ the“real-time” detection of incorporation events, where the act ofincorporation gives rise to a signaling event that can be detected. Inparticularly elegant methods, labeling components are coupled toportions of the nucleotides that are removed during the incorporationevent, eliminating any need to remove such labeling components beforethe next nucleotide is added (See, e.g., Eid, J. et al., Science,323(5910), 133-138 (2009)).

In many of the enzyme mediated template-dependent sequencing methods,the photostability of the system is important. For example, influorescent based single molecule, real time sequencing, the enzyme isexposed to excitation radiation while the sequencing reaction isoccurring. If the enzyme becomes damaged due to such irradiation, thesequencing reaction can become compromised or end.

The present invention provides methods, systems and compositions thatprovide for increased performance of such polymerization basedsequencing methods, including systems having improved photostability,among other benefits.

BRIEF SUMMARY OF THE INVENTION

In some aspects, the invention provides a polymerase enzyme substratecomprising: a protein comprising at least 60 amino acids; a nucleotideunit comprising at least one nucleoside polyphosphate attached throughits phosphate portion to a first position on the protein; a dyecomponent comprising at least one fluorescent dye moiety attached to asecond position on the protein, wherein the first and second attachmentpoints are spaced apart by a distance such that when a nucleosidephosphate attached to the protein is in the active site of thepolymerase enzyme, a fluorescent dye moiety attached to the protein isshielded by the protein from coming into contact with the polymeraseenzyme.

The substrate can be held together by covalent attachments. The proteincan comprise 60 to 1,000 amino acids. The protein can comprise 80 to 600amino acids.

The nucleotide component and dye component can be covalently attached tothe protein.

The nucleotide component can comprise two or more nucleoside phosphates.The substrate can have 2, 3, 4, 5, 6, 7, or 8 nucleotide phosphates. Thedye component can comprise two or more fluorescent dye moieties. Thesubstrate has 2, 3, or 4 fluorescent dye moieties.

The covalent attachment can be through a cysteine or lysine residue onthe protein.

The protein can have two or more nucleotide components. The protein canhave two or more dye components. The protein can have two or morenucleotide components and two or more dye components wherein when anynucleotide phosphate in a nucleotide component is in the active site ofthe polymerase enzyme, all fluorescent dye moieties in the dyecomponents are shielded by the protein from coming into contact with thepolymerase enzyme.

The protein can comprise a first protein and a second protein, the firstprotein and second protein are associated or connected, the firstprotein having one or more nucleotide components attached to it, and thesecond protein having one or more dye components attached to it. Thefirst protein and second protein can be associated. The first proteinand second protein can comprise barnase and barstar.

The first protein and second protein can be connected by a covalentlinkage. The first protein and second protein can be connected throughone or more linkers.

In some aspects, the invention provides a polymerase enzyme substratecomprising: a protein comprising at least 60 amino acids; a nucleotidecomponent comprising at least one nucleoside polyphosphate attachedthrough its phosphate portion to a first position on the protein; a dyecomponent comprising at least one fluorescent dye moiety attached to asecond position on the protein, wherein the first and second attachmentpoints are spaced apart by a distance of greater than 2 nm.

The protein can have two or more nucleotide components. The protein canhave two or more dye components. The protein can have two or morenucleotide components and two or more dye components wherein when anynucleotide phosphate in a nucleotide component is in the active site ofthe polymerase enzyme, all fluorescent dye moieties in the dyecomponents are shielded by the protein from coming into contact with thepolymerase enzyme.

In some aspects, the invention provides a method for nucleic acidsequencing comprising: providing an array of individually observableenzyme-nucleic acid template complexes on a chip; exposing the chip tothe reagents for polymerase mediated nucleic acid synthesis of a growingnucleic acid strand, the reagents comprising a plurality of differentlylabeled polymerase enzyme substrates including at least one proteinshield nucleotide; and optically monitoring the incorporation of thenucleoside monophosphate portions of the differently labeled polymeraseenzyme substrates into the growing nucleic acid strand over time,thereby determining the sequence of at least a portion of the nucleicacid template.

In some aspects, the invention provides a polymerase enzyme substratecomprising: a nucleotide component comprising at least one nucleosidepolyphosphate attached through its phosphate portion to a protein,wherein the protein has a plurality of labels embedded within it,whereby, when the polymerase enzyme substrate associates with apolymerase enzyme, the labels within the protein do not come intocontact with a polymerase enzyme.

In some aspects, the invention provides the use of a protein sequencecomprising at least 60 amino acids attached to and positioned between anucleotide component and a dye component for inhibiting contact betweensaid label and a polymerase enzyme during a polymerase catalysed nucleicacid synthesis reaction incorporating a nucleotide moiety of saidnucleotide component.

In some aspects, the invention provides a method of shielding apolymerase enzyme when used in nucleic acid synthesis from labelednucleotides, comprising providing a protein of at least 60 amino acids,having at least 20 amino acids in the primary sequence between theattachment point of each nucleotide and its respective label.

In some aspects, the invention provides a labeled nucleotide analogcomprising: an avidin protein having four subunits, each subunitcomprising one biotin binding site; one or two nucleotide componentseach comprising one or more phospholinked nucleotide moieties; and oneor two dye components each comprising one or more dye moieties; whereineach component is bound to the avidin protein through a biotin moietyattached to a binding site on the avidin protein; and wherein at leastone of the nucleotide or dye components comprise a bis-biotin moietybound to two of the biotin binding sites on the avidin protein.

The nucleotide component can comprise a bis-biotin moiety, and thelabeled nucleotide analog has two dye components each bound to theavidin through a single biotin moiety. The dye component can comprise abis-biotin moiety, and the labeled nucleotide analog has two nucleotidecomponents each bound to the avidin through a single biotin moiety. Thelabeled nucleotide analog can have one dye component and one nucleotidecomponent and each of the dye component and the nucleotide component cancomprise a bis-biotin moiety.

The number of dye moieties can be between 1 and 18 and the number ofnucleotide moieties can be between 1 and 18. The number of dye moietiescan be 1, 2, or 3 and the number of nucleotide moieties can be 6, 7, or8. The number of bonds between biotins on the bis-biotin moiety can bebetween 15 and 50. The avidin protein can comprise either streptavidinor tamavidin. The dye moieties can comprise fluorescent labels.

In some aspects, the invention provides a reaction mixture forsequencing a nucleic acid template comprising: a polymerase enzymecomplex comprising a polymerase enzyme, a template nucleic acid, andoptionally a primer hybridized to the template nucleic acid, wherein thepolymerase enzyme complex is immobilized on a surface; and sequencingreagents in contact with the surface comprising reagents for carryingout nucleic acid synthesis including 2 or more types of labelednucleotide analogs, wherein one or more of the types of nucleotideanalog is a protein-shielded nucleotide analog comprising an avidinprotein having four subunits, each subunit comprising one biotin bindingsite; one or two nucleotide components each comprising one or morephospholinked nucleotide moieties; and one or two dye components eachcomprising one or more dye moieties; wherein each component is bound tothe avidin through a biotin moiety attached to a binding site on theavidin protein; and wherein at least one of the nucleotide or dyecomponents comprise a bis-biotin moiety bound to two of the biotinbinding sites on the avidin protein.

The nucleotide component can comprise a bis-biotin moiety, and a labelednucleotide analog has two dye components each bound to the avidinthrough a single biotin moiety. The dye component can comprise abis-biotin moiety, and a labeled nucleotide analog has two nucleotidecomponents each bound to the avidin through a single biotin moiety.

A labeled nucleotide analog can have one dye component and onenucleotide component and each of the dye component and the nucleotidecomponent can comprise a bis-biotin moiety. The number of dye moietiescan be between 1 and 18 and the number of nucleotide moieties can bebetween 1 and 18 for each nucleotide analog. The number of dye moietiescan be 1, 2, or 3 and the number of nucleotide moieties can be 6, 7, or8 for each nucleotide analog. The number of bonds between biotins on abis-biotin moiety can be between 15 and 50. The avidin protein cancomprise either streptavidin or tamavidin. The dye moieties can comprisefluorescent labels.

In some aspects, the invention provides a method for sequencing anucleic acid template comprising: providing a polymerase enzyme complexcomprising a polymerase enzyme, a template nucleic acid, and optionallya primer hybridized to the template nucleic acid, wherein the polymeraseenzyme complex is immobilized on a surface; adding sequencing reagentsin contact with the surface comprising reagents for carrying out nucleicacid synthesis including 2 or more types of labeled nucleotide analogs,wherein one or more of the types of nucleotide analog is aprotein-shielded nucleotide analog comprising; an avidin protein havingfour subunits, each subunit comprising one biotin binding site; one ortwo nucleotide components each comprising one or more phospholinkednucleotide moieties; and one or two dye components each comprising oneor more dye moieties; wherein each component is bound to the avidinthrough a biotin moiety attached to a binding site on the avidinprotein; and wherein at least one of the nucleotide or dye componentscomprise a bis-biotin moiety bound to two of the biotin binding sites onthe avidin protein; and determining the sequential addition ofnucleotides to a nucleic acid strand complementary to a strand of thetemplate nucleic acid by observing the interaction of the labelednucleotide analogs with the polymerase enzyme complex.

The nucleotide component can comprise a bis-biotin moiety, and a labelednucleotide analog can have two dye components each bound to the avidinthrough a single biotin moiety. The dye component can comprise abis-biotin moiety, and a labeled nucleotide analog can have twonucleotide components each bound to the avidin through a single biotinmoiety. A labeled nucleotide analog can have one dye component and onenucleotide component and each of the dye component and the nucleotidecomponent can comprise a bis-biotin moiety.

The number of dye moieties can be between 1 and 18 and the number ofnucleotide moieties can be between 1 and 18 in each nucleotide analog.The number of dye moieties can be 1, 2, or 3 and the number ofnucleotide moieties can be 6, 7, or 8 in each nucleotide analog. Thenumber of bonds between biotins on a bis-biotin moiety can be between 15and 50. The avidin protein in an analog can comprise either streptavidinor tamavidin. The dye moieties can comprise fluorescent labels.

In some aspects, the invention provides a system for sequencing nucleicacids comprising: a chip comprising a plurality of polymerase enzymecomplexes bound thereto, each polymerase enzyme complex individuallyoptically resolvable, each polymerase enzyme complex comprising apolymerase enzyme, a template nucleic acid, and optionally a primerhybridized to the template nucleic acid, sequencing reagents in contactwith the surface comprising reagents for carrying out nucleic acidsynthesis including 2 or more types of fluorescently labeled nucleotideanalogs, wherein one or more of the types of nucleotide analog is aprotein-shielded nucleotide analog comprising; an avidin protein havingfour subunits, each subunit comprising one biotin binding site; one ortwo nucleotide components each comprising one or more phospholinkednucleotide moieties; and one or two dye components each comprising oneor more dye moieties; wherein each component is bound to the avidinthrough a biotin moiety attached to a binding site on the avidinprotein; and wherein at least one of the nucleotide or dye componentscomprise a bis-biotin moiety bound to two of the biotin binding sites onthe avidin protein; and an illumination system for illuminating thepolymerase enzyme complexes; and an optical detection system fordetecting fluorescence from the labeled nucleotide analogs while theyare interacting with the polymerase enzyme complexes; and a computer foranalyzing the signals detected by the detection system to determine thesequential addition of nucleotides to a nucleic acid strandcomplementary to a strand of the template nucleic acid.

The nucleotide component can comprise a bis-biotin moiety, and a labelednucleotide analog has two dye components each bound to the avidinthrough a single biotin moiety. The dye component can comprise abis-biotin moiety, and a labeled nucleotide analog has two nucleotidecomponents each bound to the avidin through a single biotin moiety. Thelabeled nucleotide analog can have one dye component and one nucleotidecomponent and each of the dye component and the nucleotide component cancomprise a bis-biotin moiety.

The number of dye moieties in the nucleotide analogs can be between 1and 18 and the number of nucleotide moieties can be between 1 and 18 ineach nucleotide analog. The number of dye moieties can be 1, 2, or 3 andthe number of nucleotide moieties can be 6, 7, or 8 in each nucleotideanalog. The number of bonds between biotins on a bis-biotin moiety canbe between 15 and 50. The avidin protein in a nucleotide analog cancomprise either streptavidin or tamavidin. The dye moieties can comprisefluorescent labels.

In some aspects, the invention provides a labeled nucleotide analogcomprising: two avidin proteins connected to each other through acompound comprising two bis-biotin moieties and comprising a dyecomponent having one or more dye moieties, wherein one of the avidins isattached to a bis-biotin moiety comprising a first nucleotide componenthaving one or more phospholinked nucleotides, and wherein the otheravidin is attached to a bis-biotin moiety comprising a second nucleotidecomponent having one or more phospholinked nucleotides.

The first nucleotide component and second nucleotide component can bethe same.

The number of dye moieties can be between 1 and 18 and the number ofnucleotide moieties can be between 1 and 18.

The number of dye moieties can be 1, 2, or 3 and the number ofnucleotide moieties can be between 8 and 24. The number of bonds betweenbiotins on the bis-biotin moiety can be between 15 and 50. The avidinproteins can comprise either streptavidin or tamavidin. The dye moietiescan comprise fluorescent labels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) illustrates single molecule real time sequencing with aconventional nucleotide analog, FIG. 1 (B) illustrates single moleculereal time sequencing with a nucleotide analog comprising a proteinshield.

FIG. 2 shows various constructs for nucleotide analogs of the inventionhaving a protein shield. FIG. 2(A) shows a nucleotide analog having onenucleotide moiety and one fluorescent moiety. FIG. 2(B) shows anucleotide analog having on nucleotide moiety and two fluorescentmoieties. FIG. 2(C) shows a nucleotide analog having two nucleotidemoieties and one dye moiety. FIG. 2(D) shows a nucleotide analog of theinvention having two fluorescent moieties and two nucleotide moieties,each moiety attached at a different place on the protein. FIG. 2(E)shows a nucleotide analog of the invention having two fluorescentmoieties and three nucleotide moieties, each moiety attached at adifferent place on the protein. FIG. 2(F) shows a nucleotide analog ofthe invention having three fluorescent moieties and two nucleotidemoieties, each moiety attached at a different place on the protein. FIG.2(G) shows a nucleotide analog of the invention having three fluorescentmoieties and three nucleotide moieties, each moiety attached at adifferent place on the protein. FIG. 2(H) shows a nucleotide analog ofthe invention having two fluorescent moieties and one nucleotidemoieties, the two fluorescent moieties connected to the same attachmentpoint on the protein. FIG. 2(I) shows a nucleotide analog of theinvention having two fluorescent moieties and two nucleotide moieties,the two fluorescent moieties connected to the same attachment point onthe protein, and the two nucleotide moieties connected to a secondattachment point on the protein. FIG. 2(J) shows a nucleotide analog ofthe invention having two fluorescent moieties and two nucleotidemoieties, the two fluorescent moieties connected to the same attachmentpoint on the protein, and the two nucleotide moieties are each connectedto a different attachment point on the protein. FIG. 2(K) shows anucleotide analog of the invention having three fluorescent moieties andtwo nucleotide moieties, the three fluorescent moieties connected to thesame attachment point on the protein, and the two nucleotide moietiesconnected to a second attachment point on the protein. FIG. 2(L) shows anucleotide analog of the invention having four fluorescent moieties andtwo nucleotide moieties, two of the fluorescent moieties are connectedto one attachment point on the protein, the other two fluorescentmoieties are connected to a second attachment point, and the twonucleotide moieties connected to a third attachment point on theprotein.

FIG. 3 shows a three dimensional representation of a Ubiquitin proteinfor use as a protein shield.

FIG. 4 shows a three dimensional representation of a TOP7 protein foruse as a protein shield.

FIG. 5 shows a three dimensional representation of a Tamavidin proteinfor use as a protein shield.

FIG. 6 shows a three dimensional representation of a Papain protein foruse as a protein shield.

FIG. 7 shows a three dimensional representation of a Maltose BindingProtein for use as a protein shield.

FIG. 8 shows a three dimensional representation of Barnase/Barstar foruse as a protein shield.

FIG. 9 shows a three dimensional representation of SNAP tag protein foruse as a protein shield.

FIG. 10 shows a three dimensional representation of Beta lactamase foruse as a protein shield.

FIG. 11 shows a three dimensional representation of a Coiled-Coil Domainfor use as a protein shield.

FIG. 12 shows a three dimensional representation of Allophycocyanin foruse as a nucleotide analog having buried chromophores.

FIG. 13 shows a three dimensional representation of Green FluorescentProtein for use as a nucleotide analog having buried chromophores.

FIG. 14 shows nucleotide analogs of the invention comprising abis-biotin moiety in (A) the dye component, (B) the nucleotidecomponent, or (C) both the dye component and nucleotide component.

FIG. 15 shows a protein shield nucleotide analog of the invention havingan avidin protein connected to a bis-biotin dye component and to twobiotin dinucleotide components. Also shown in FIG. 15 are the bis-biotindye component and biotin dinucleotide component. FIG. 15 also includestwo exemplary trifunctional linkers T1 and T7 that can be used toproduce the bis-biotin dye component and biotin nucleotide components.

FIGS. 16(A) and 16(B) each show exemplary bis-biotin moieties.

FIG. 17 shows a protein shield nucleotide analog of the invention withan avidin attached to a bis-biotin dye component having two dyes. Alsoshown is an exemplary bis-biotin double-dye component.

FIG. 18 shows an exemplary synthesis of a bis-biotin double-dye.

FIG. 19 shows a protein shield nucleotide analog of the invention havingtwo nucleotide components and eight phospholinked nucleotides. Alsoshown is the trifunctional linker Sh and an Sh alternative.

FIG. 20 shows an exemplary nucleotide component having fourphospholinked nucleotides attached to one biotin.

FIG. 21 shows a protein shield nucleotide analog of the invention havingtwo nucleotide components and 12 phospholinked nucleotides.

FIG. 22 shows an exemplary nucleotide component with one biotin and sixphospholinked nucleotides.

FIG. 23 shows an exemplary protein shield nucleotide analog of theinvention having a bis-biotin dye component with two dyes and abis-biotin component having six phospholinked nucleotides. The figurealso shows how this protein shield nucleotide analog can be made usingstreptavidin, a bis-biotin dye component, and a bis-biotinhexanucleotide component.

FIG. 24 shows an exemplary bis-biotin hexanucleotide component.

FIG. 25 shows components G (Gal), T, Chx, T12, Biotin-X, Aba, 6C, anddT6P that can be used to prepare a bis-biotin hexanucleotide component.

FIG. 26 shows an exemplary synthesis of a bis-biotin hexanucleotidecomponent.

FIG. 27 (A) shows a protein shield protein shield nucleotide analog ofthe invention having two avidins held together through a tetra-biotinmoiety made of two bis-biotins and having a dye component. Each avidinprotein has a nucleotide component connected through a bis-biotinmoiety.

FIG. 27(B) shows an exemplary protein shield nucleotide of the inventionwhere the central tetra-biotin moiety has a dye component with two dyes,and each streptavidin has a nucleotide component with six phospholinkednucleotide moieties attached through a bis-biotin moiety. FIG. 27(B)also illustrates that this compound can be formed from a twostreptavidins, a tetra-biotin dye component, and two bis-biotinhexanucleotide components.

FIG. 28 shows an exemplary tetra-biotin dye component having two dyes,one being a FRET donor, and the other being a FRET acceptor. The figurealso shows T8, an exemplary trifunctional linker that can be used tomake such a tetra-biotin dye component.

FIG. 29 shows a schematic showing one way of carrying out real-timesingle molecule nucleic acid sequencing. FIG. 29(I) shows the sequencingprocess involving observing a polymerase within an observation volume,FIG. 29(II) shows an exemplary plot of signal intensity versus timeduring the sequencing process.

FIG. 30 shows a system for carrying out real-time single moleculesequencing.

FIG. 31 shows how having sets of nucleotide analogs with multipleintensity levels can allow for calling pulses that would otherwise bemerged. The pulse train shown in FIG. 31(A) illustrates a run of threeGs having a short inter pulse distance (IPD) between them. FIG. 31(B)illustrates a pulse train with similar overall kinetics to that in FIG.31(A), but with the use of multi-level dyes.

FIG. 32 shows one construct of a nucleotide analog having a streptavidinprotein shield.

FIG. 33 shows a construct of a nucleotide analog having a streptavidinprotein shield that was used in real-time single molecule sequencing.

FIG. 34 shows histograms of read lengths for a control (A) and for asequencing reaction with a nucleotide analog having a protein shieldshowing improved photostability (B).

FIG. 35 shows read length plots for four movies of a control (A) and fora sequencing reaction with a nucleotide analog having a protein shieldshowing improved photostability (B).

FIG. 36 shows an exemplary bis-biotin compound having six phospholinkednucleotides and having four kinetic modifier groups.

DETAILED DESCRIPTION OF THE INVENTION

When described, in what follows, are certain features of the inventionwhere a list of potential choices can and have been set out below, seefor example the number of dyes moieties and nucleotide moieties in anucleotide analog, the reader is instructed to understand that this ispurely in the interests of conciseness and that any one of the exemplaryfeatures in any one of said paragraphs can be combined with any other ofthe exemplary features in any other of said paragraphs in anycombination the skilled person chooses. This specification is to beconstrued accordingly.

In single-molecule real-time sequencing using fluorescence detection,the enzyme is illuminated with excitation light while a sequencingreaction is taking place. In some cases, the illumination results inphotodamage which damages or kills the polymerase enzyme. This damagecan cause the sequencing reaction to end, resulting in shorter readlengths than desired. The inventors have performed experiments whichdemonstrate that significantly longer read lengths can in some cases beobtained in the dark than can be obtained for the same sequencingreaction under illumination. The inventors have also performedexperiments which indicate that damage to the enzyme under illuminationcan be accompanied by the formation of a covalent bond between afluorescent dye moiety on a nucleotide analog and the polymerase enzyme.Thus, it is believed that the stability of the enzyme can be compromisedwhen there is contact between the enzyme and a fluorescent moiety on anucleotide analog which is in the active site of the enzyme. In somecases, it appears that this mechanism constitutes the dominant mode ofdegradation.

The inventors have found that photodamage can be mitigated andsequencing readlengths improved by incorporation of a shielding proteininto a nucleotide analog. The nucleotide analog is constructed such thatthe shielding protein is disposed between the nucleotide phosphateportion and the fluorescent dye portion of the nucleotide analog. Thesize and position of the protein are chosen such that the fluorescentdye portion of the analog does not come into contact with the polymeraseenzyme when the nucleotide portion is held within the active site of thepolymerase. Preventing contact between the fluorescent dye and thepolymerase prevents the formation of a covalent bond to the polymerase.By shielding the enzyme from contact with the fluorescent dye, theprotein blocks a significant photodamage pathway, resulting in longerenzyme life under illumination.

The nucleotide (nucleoside phosphate) portion of the analog is attachedto the shielding protein through the polyphosphate portion of thenucleotide. With this type of attachment, when the nucleotidemonophosphate portion of the nucleotide analog is incorporated into thegrowing nucleic acid strand, the portion of the nucleotide analog havingthe shielding protein and the fluorescent dye is cleaved from theportion of the nucleotide that gets cleaved, and it diffuses away toallow for incorporation of the next nucleotide into the chain withoutinterference with these moieties.

FIG. 1 shows a schematic illustration of the incorporation of ashielding protein into a nucleotide analog. FIG. 1A illustrates realtime single molecule sequencing with a conventional nucleotide analog.The polymerase enzyme 110 is bound to the surface of a substrate, suchas a chip, 160, for example with a linker 150. The polymerase enzyme 110is complexed with a template nucleic acid having a template strand 120and a primer/growing strand 130. Sequencing is performed by observingthe enzyme while it is incorporating nucleotides into the growingstrand. The nucleotide analog that gets incorporated into the growingstrand generally spends more time in the active site than a nucleotideanalog that does not get incorporated, allowing for the identificationof the incorporated nucleotide. When an incorporation takes place, thenucleoside monophosphate portion of the nucleotide analog isincorporated into the growing chain, and the remainder of the nucleotideanalog including the fluorescent label is cleaved away and the enzyme isready for the next incorporation. By watching the incorporation of basesover time, the sequence of the template nucleic acid 120 can bedetermined by determining the series of nucleotides that are incorporateinto the growing strand 130. In the embodiment described here, thepolymerase enzyme complex is tethered to a substrate. It can beadvantageous for the polymerase enzyme complex to remain in one discreteregion of space over the sequencing reaction to assist in opticallyobserving the complex over time. While here, the complex is described asattached to a substrate surface, there are other ways to providerestricting the movement of the polymerase complex such as the use ofgels, or the use of discrete volumes.

FIG. 1(A) shows a conventional nucleotide analog 140, held in the enzymeprior to incorporation. The nucleotide portion of the analog 144 is heldin the active site of the enzyme in position for incorporation. FIG.1(A)(2) illustrates that while the nucleotide analog is associated withthe enzyme, the fluorescent moiety 142 can come into contact with theenzyme. During this process, the enzyme is illuminated with excitationlight to allow for observation of the fluorescent moiety. When thefluorescent moiety absorbs the excitation light, it enters into anexcited state. It is believed that having the excited fluorescent moietyin the vicinity of the enzyme, and in particular in when the fluorescentmoiety comes into contact with the enzyme, damage to the enzyme canresult, adversely affecting the sequencing reaction. The damage to theenzyme can result in slowing the enzyme, altering its effectiveness, orin completely halting the polymerase reaction. Applicants note that insome cases the term substrate is used to refer to an “enzyme substrate”such as a nucleotide analog, and that in some cases, the term substrateis used to refer to a solid support. Which type of “substrate” is beingreferred to will be clear from the context of the use of the term andshould be understood by one of ordinary skill in the art.

FIG. 1(B) shows a nucleotide analog or polymerase enzyme substrate ofthe invention 170 having a shielding protein 176. Sequencing can becarried out with analogs having shielding proteins by the same processesdescribed above for conventional nucleotide analogs. As with aconventional enzyme substrate, the nucleotide portion 174 of the analogis held in the active site of the enzyme prior to incorporation. Thenucleotide analog of the invention has a nucleotide portion 174connected to the shielding protein through the phosphate portion of thenucleotide. In some cases, the nucleotide is attached to the proteinthrough a linker group 179. A fluorescent moiety is attached to theshielding protein, in some eases through a linking group 178. FIG.1(B)(2) illustrates that the shielding protein 176 prevents contactbetween the polymerase enzyme and the fluorescent dye moiety 172. Byshielding the enzyme from contact with the fluorescent dye moiety whilethe nucleotide portion of the analog is in the active site of theenzyme, the enzyme is protected from photodamage due to contact with thefluorescent moiety's excited state. While the usefulness of thenucleotide analogs of the invention is illustrated with the descriptionabove of SMRT sequencing, it is to be understood that these analogs canbe used with any suitable sequencing method.

There are several structural features that can be used to inhibit orprevent the florescent dye from coming into contact with the polymeraseenzyme when the nucleotide portion of the nucleic acid analog is in theactive site. One feature is the size of the shielding protein. Theshielding protein generally has greater than about 60 amino acids. Theshielding protein can be greater than about 80 amino acids. Theshielding protein can be greater than about 200 amino acids. In somecases the shielding protein has from about 60 amino acids to about 2,000amino acids. In some cases it has from about 60 amino acids to about1,000 amino acids. In some cases it has from about 80 amino acids toabout 600 amino acids.

Another feature that can be used to prevent contact between thefluorescent moiety and the polymerase enzyme is the distance between thepoints of attachment of the nucleotide portion and the fluorescent dyeportion of the nucleotide analog. The attachment point for thefluorescent moiety and the nucleotide are generally distal from eachother on the enzyme. In some cases, the attachment points are 2 nm apartor greater. In some cases the attachment point is 4 nm apart or greater.The distance between the attachment points can be either through space,or can be the distance across the surface of the protein. The distancethrough space can be determined by modeling the three dimensionalstructure of the protein. Current modeling software can accuratelydescribe the 3 dimensional structure of proteins. In some cases, thesemodels can be informed by X-ray crystal structure and/or X-ray orneutron scattering to improve the accuracy. In some cases, the distancebetween the attachment point of the nucleotide and the attachment pointof the fluorescent moiety is greater than one quarter of a distancearound the protein. In some cases, the distance between the attachmentpoints is greater than a third of a distance around the protein. Adistance around the protein can be determined, for example by obtaininga structure of the protein, treating the structure of the protein as anellipsoid, and tracing an ellipse around the ellipsoid including thepoints of attachment.

In accordance with the shielding aspects of the invention, it isdesirable to have the attachment points of a nucleotide component and adye component on the protein be spaced apart. This spacing can bedescribed by a three-dimensional (through space) distance betweenattachment points, or by a distance over the surface of a proteinbetween attachment points. Another way of characterizing the distancebetween the attachment points is in terms of linear distance betweenattachment points on the primary sequence of the protein. We have found,for example, that it is typically desirable that the linkages be atleast about 20 amino acid units apart for a protein of at least 60 aminoacids. In some cases it is desirable that the linkages be at least about30 amino acids apart in the primary sequence.

Another feature that can be used to prevent contact between thefluorescent moiety and the polymerase enzyme is the length and theflexibility of a linker between the fluorescent moiety and the proteinand a linker between the nucleotide and the protein. Such linkers arenot required but can be used to attach the various portions of thenucleotide analog. FIG. 1(B) shows linker 178 between the fluorescentmoiety 172 and the shielding protein, and optional linker 179between thenucleotide and the shielding protein. The linkers can comprise anysuitable subunits. Examples of linkers are provided in more detailherein. The length of the linker is chosen such that, taking intoaccount the shortest distance between attachment points on the shieldingprotein, that the dye will be prevented from contacting the polymeraseenzyme when the nucleotide moiety is in the active site of the enzyme.

One of skill in the art is able to determine that a particular structurewill prevent contact of the fluorescent dye with the polymerase enzyme.For example, computer based or physical molecular models can beconstructed that describe the extent of movement of a particular moietywithin an ensemble of molecules. In some cases, knowledge of a proteinstructure, for example from x-ray crystallography combined with aknowledge of molecular dimensions of sub-structures can be combined todetermine whether contact between the fluorescent dye and the polymeraseenzyme will occur. As used in this context, preventing contact betweenthe fluorescent dye and the polymerase enzyme means that under theconditions of the sequencing reaction, contact will occur, if at all,only very rarely, to the extent that such contact would not lead todiscernable photodamage events.

We have found that in some cases it is advantageous to providenucleotide analog constructs having more than one fluorescent moietyand/or more than one nucleotide moiety. For example, having multiple dyemoieties. In some cases the polymerase enzyme substrate or nucleotideanalog has 1 to 10 fluorescent dye moieties. In some eases the analoghas 1 to 4 fluorescent dye moieties. In some cases the analog has 1, 2,3, 4, 5, 6, 7, or 8 fluorescent dye moieties. In some cases the analoghas at least 1, 2, 3, 4, 5, 6, 7, or 8 fluorescent dye moieties. In somecases the polymerase enzyme substrate or nucleotide analog has 1 to 10fluorescent nucleotide moieties, in some cases the analog has 1 to 4nucleotide moieties. In some cases the analog has 1, 2, 3, 4, 5, 6, 7,or 8 nucleotide moieties. The nucleotide analog of the invention canhave any suitable combination of 1 to 10 fluorescent dye moieties and 1to 10 nucleotide moieties.

In some cases, each of the moieties is attached to a differentattachment point on the protein. In some cases, an attachment point onthe shielding protein branches out to have multiple dyes or multiplenucleotides. In some cases, a single dye or single nucleotide can havemultiple attachment points. Any suitable combination of dyes andattachment points, or nucleotides and attachment points can be used.

FIG. 2 shows various exemplary nucleotide analogs of the invention. Ineach case, the nucleotide moieties 230 are represented by triangles, andthe fluorescent dye moieties 210 are represented by hexagons. In eachcase the moieties are attached to a shielding protein 220. The moietiescan be attached using linkers or they can be attached directly to theproteins. Generally, covalent attachment of the moieties is preferred,but in some cases, the affinity binding of a protein can be used forattachment of one or more of the moieties. For example, in some cases, aprotein such as avidin or streptavidin can be used, and the protein'saffinity for biotin can be used to attach a moiety to the protein. FIG.2(A) shows a nucleotide analog having one nucleotide moiety and onefluorescent moiety. FIG. 2(B) shows a nucleotide analog having onnucleotide moiety and two fluorescent moieties. Multiple dyes can resulta brighter analog. In addition, different dyes can be used to providedifferent color combinations. In addition dyes which have FRETinteractions can be used to provide higher brightness, and a largerStokes shift (wavelength difference between excitation and emission).FIG. 2(C) shows a nucleotide analog having two nucleotide moieties andone dye moiety. Having multiple nucleotide analogs can be used to raisethe effective concentration of nucleotide. For instance having a highernumber of nucleotide moieties can result in a faster reaction rate at agiven concentration of analog, or can be used to produce the samereaction rate at a lower concentration of analog. FIGS. 2 (D) through2(L) provide various combinations of nucleotide moieties and fluorescentdye moieties that allow for optimizing the levels of brightness andeffective concentration. In some cases each of the moieties is attacheddirectly to the shielding protein. In some cases, an attachment pointwill be linked to multiple moieties. FIG. 2(D) shows a nucleotide analogof the invention having two fluorescent moieties and two nucleotidemoieties, each moiety attached at a different place on the protein. FIG.2(E) shows a nucleotide analog of the invention having two fluorescentmoieties and three nucleotide moieties, each moiety attached at adifferent place on the protein. FIG. 2(F) shows a nucleotide analog ofthe invention having three fluorescent moieties and two nucleotidemoieties, each moiety attached at a different place on the protein. FIG.2(G) shows a nucleotide analog of the invention having three fluorescentmoieties and three nucleotide moieties, each moiety attached at adifferent place on the protein. FIG. 2(H) shows a nucleotide analog ofthe invention having two fluorescent moieties and one nucleotidemoieties, the two fluorescent moieties connected to the same attachmentpoint on the protein. FIG. 2(I) shows a nucleotide analog of theinvention having two fluorescent moieties and two nucleotide moieties,the two fluorescent moieties connected to the same attachment point onthe protein, and the two nucleotide moieties connected to a secondattachment point on the protein. FIG. 2(J) shows a nucleotide analog ofthe invention having two fluorescent moieties and two nucleotidemoieties, the two fluorescent moieties connected to the same attachmentpoint on the protein, and the two nucleotide moieties are each connectedto a different attachment point on the protein. FIG. 2(K) shows anucleotide analog of the invention having three fluorescent moieties andtwo nucleotide moieties, the three fluorescent moieties connected to thesame attachment point on the protein, and the two nucleotide moietiesconnected to a second attachment point on the protein. FIG. 2(L) shows anucleotide analog of the invention having four fluorescent moieties andtwo nucleotide moieties, two of the fluorescent moieties are connectedto one attachment point on the protein, the other two fluorescentmoieties are connected to a second attachment point, and the twonucleotide moieties connected to a third attachment point on theprotein. It will be understood from the description and the drawingsthat there are many other combinations of multiple fluorescent moietiesand nucleotide moieties that can be used with the instant invention. Inaddition to compounds of the invention having multiple moieties attachedto a single attachment point on a shielding protein, in some cases, asingle moiety can be attached to the protein shield through multipleattachment points.

The attachment of the nucleotide and fluorescent moieties to theshielding proteins can be by any suitable means. In preferredembodiments, the moieties are covalently linked to the proteins. Whilein some cases affinity pair linkages can be useful in the invention, wehave found that covalent linkages are often preferred due to theirstability and consistency. In sequencing systems, there will often befour different nucleotide analogs, each having one of four bases (e.g.A, G, C, T, or A, G, C, U). For consistent sequencing results, it isdesired to have a set of nucleotide analogs that can be readilypurified, for which quality control experiments can be readilyperformed, and which will be stable over time without dissociation orrearrangement. Covalently linked structures can meet these criteria.

Covalent linkage of moieties to proteins is well known in the art. Thereactive groups on various amino acids can be used to provide specificsites of attachment. Reactive groups for the attachment of moieties tothe protein include amine groups on lysine or arginine, the thiol groupon cysteine, the acid group on aspartic acid or glutamic acid, and thehydroxyl group on serene or threonine. In some cases, an availableprotein will have appropriate residues for connection of the moieties.In other cases, the appropriate residues can be engineered into theprotein. Using genetic engineering to produce a desired protein havingvarious amino acids removed or added is a common and well understoodpractice.

The different reactivity of different groups on the protein can be usedto direct specific moieties to different attachment points on theprotein. For example, a nucleotide moiety can be connected to a specificcysteine at one desired attachment point, and a fluorescent moiety canbe attached to a lysine at a second attachment point. In some cases, thesame type of residue will have different reactivity due to where itresides on the protein, allowing selective attachment. For example, aprotein may have three lysine moieties where each has a differentreactivity. Attachment can be carried out such that only the mostreactive lysine is modified, or alternatively, attachment can be carriedout by protecting the two most reactive lysines, then reacting themoiety of interest with the third, least reactive lysine.

There are many types of chemical reactions that can be used to reactwith specific amino acid residues on proteins. For example, couplingthrough the cysteine thiol can be accomplished using a reaction withmaleimide. Cysteine groups can also be coupled with allylic halides,phenylmethyl halides, alkyl halides, or alpha-halo carbonyl groups.Amine groups can be coupled to activated carboxylates or activatedsulfonic acids. Amine or carboxylate functionality on the protein can beused to produce amide linkages. Linkages containing nitrogen doublebonds such as oxime or hydrazones can be used. Highly selective linkagescan be formed using cycloaddition chemistry such as the Huisgen1,3-dipolar azide-alkyne cycloaddition. See e.g. Advances inBioconjugation, Kalia, J, Raines, R. T., Curr Org. Chem. 2010 January;14(2): 138-147, Besanceney-Webler et al., “Increasing the Efficacy ofBioorthogonal Click Reactions for Bioconjugation” Angew. Chem. Int. Ed.2011, 50, 8051-8056, and DiMarco et al. International Journal ofNanornedicine, December 2009, 37-49.

The moieties can be attached to the shielding protein through unnaturalamino acids that are introduced into the protein, allowing for specificattachment chemistry. See, for example, the work of Peter Schultz, e.g.Noren et al., “A general method for site-specific incorporation ofunnatural amino acids into proteins”, Science, 244:182-188, 1989, andEllman et at “Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins”, Methods in Enzymology, Volume 202,1991, Pages 301-336.

Many other methods of chemically modifying proteins are known in theart. See e.g. “Chemical modification of proteins at cysteine:opportunities in chemistry and biology” Chalker J M, Bernardes G J, LinY A, Davis B G, Chem Asian J. 2009 May 4; 4(5):630-40, “Chemoselectiveligation and modification strategies for peptides and proteins”Hackenberger C P, Schwarzer D. Angew Chem Int Ed Engl. 2008;47(52):10030-74, “Chemoselective modification of proteins: hitting thetarget”, Carrico I S, Chem Sac Rev. 2008 July; 37(7):1423-31,“Modification of tryptophan and tryptophan residues in proteins byreactive nitrogen species”, Yamakura F, Ikeda K, Nitric Oxide. 2006March; 14(2):152-61, Chemical modification of proteins, Came A F,Methods Mol. Biol. 1994; 32:311-20, Selective chemical modification ofproteins, Shaw E, Physiol Rev. 1970 April; 50(2):244-96, and “Chemicalreagents for protein modification” By Roger L. Lundblad, CRC Press,2004.

Reactive functional groups can be used to attach the moieties to theshielding proteins and to attach moieties to linkers and linkers toproteins. Reactions for this purpose and other useful reactions arediscussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed.,John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES,Academie Press, San Diego, 1996; and Feeney et at., MODIFICATION OFPROTEINS; Advances in Chemistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982.

Useful reactive functional groups include, for example:

-   (a) carboxyl groups and derivatives thereof including, but not    limited to activated esters, e.g., N-hydroxysuccinimide esters,    N-hydroxyphthalimide, N-hydroxybenztriazole esters, acid halides,    acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl,    alkynyl and aromatic esters, activating groups used in peptide    synthesis and acid halides;-   (b) hydroxyl groups, which can be converted to esters, sulfonates,    phosphoramidates, ethers, aldehydes, etc.-   (c) haloalkyl groups, wherein the halide can be displaced with a    nucleophilic group such as, for example, an amine, a carboxylate    anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting    in the covalent attachment of a new group at the site of the halogen    atom;-   (d) dienophile groups, which are capable of participating in    Diels-Alder reactions such as, for example, maleimido groups;-   (e) aldehyde or ketone groups, allowing derivatization via formation    of carbonyl derivatives, e.g., imines, hydrazones, semicarbazones or    oximes, or via such mechanisms as Grignard addition or alkyllithium    addition;-   (f) sulfonyl halide groups for reaction with amines, for example, to    form sulfonamides;-   (g) thiol groups, which can be converted to disulfides or reacted    with acyl halides, for example;-   (h) amine or sulfhydryl groups, which can be, for example, acylated,    alkylated or oxidized;-   (i) alkenes, which can undergo, for example, cycloadditions,    acylation, Michael addition, etc;-   (j) epoxides, which can react with, for example, amines and hydroxyl    compounds; and-   (k) phosphoramidites and other standard functional groups useful in    nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assembleor utilize the nucleotide analogue. Alternatively, a reactive functionalgroup can be protected from participating in the reaction by thepresence of a protecting group. Those of skill in the art understand howto protect a particular functional group such that it does not interferewith a chosen set of reaction conditions. For examples of usefulprotecting groups, see, for example, Greene et al., PROTECTIVE GROUPS INORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

The shielding proteins can be modified, for example at the C-terminaland/or N-terminal region of the protein. For example, the one or moremodifications can be a polyhistidine tag, a HIS-10 tag, a HIS-6 tag, analanine tag, an Ala10 tag, an Ala 16 tag, a biotin tag, a GST tag, aBiTag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D) tag, a lysine tag,a NanoTag, a Cmyc tag, a tag or linker comprising the amino acidsglycine and serine, a tag or linker comprising the amino acids glycine,serine, alanine and histidine, a tag or linker comprising the aminoacids glycine, arginine, lysine, glutamine and proline, a plurality ofpolyhistidine tags, a plurality of HIS-10 tags, a plurality of HIS-6tags, a plurality of alanine tags, a plurality of Ala10 tags, aplurality of Ala16 tags, a plurality of biotin tags, a plurality of GSTtags, a plurality of BiTags, a plurality of S Tags, a plurality ofSNAP-tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, aplurality of lysine tags, a plurality of NanoTags, a plurality of Cmyctags, a plurality of tags or linkers comprising the amino acids glycineand serine, a plurality of tags or linkers comprising the amino acidsglycine, serine, alanine and histidine, a plurality of tags or linkerscomprising the amino acids glycine, arginine, lysine, glutamine andproline, biotin, avidin, one or more Factor Xa sites, one or moreenterokinase sites, thrombin sites, antibodies or antibody domains,antibody fragments, antigens, receptors, receptor domains, receptorfragments, ligands, or combinations thereof.

The protein can include one or more modifiations at both the C-terminaland N-terminal regions of the polymerase, where such features at theC-terminal and N-terminal regions are optionally the same, e.g., apolyhistidine tag (e.g., a His10 tag) at both the C-terminal andN-terminal regions. Polymerases that include exogenous or heterologousfeatures at both the C-terminal and N-terminal regions optionallyinclude a B-Tag and a polyhistidine tag (e.g., a B-Tag at the N-terminalregion and a polyhistidine tag (e.g., a His-10 tag) at the C-terminalregion). Polymerases that include a B-Tag and a polyhistidine tag canfurther include a Factor Xa recognition site. Any of these modificationscan be used as sites for attachment of one or more moieties.

The shield protein can comprise the protein ubiquitin. Ubiquitin is asmall regulatory protein that has been found in almost all tissues ofeukaryotic organisms. A variety of different modifications can occur.The ubiquitin protein has about 76 amino acids and has a molecular massof about 8.5 kDa. It is highly conserved among eukaryotic species: Humanand yeast ubiquitin share 96% sequence identity. Any suitable ubiquitinprotein can be used as the shield protein or as part of a shieldprotein. For example the human ubiquitin 1 UBQ can be used as a shieldprotein by coupling nucleotides to reactive groups on the protein asdescribed herein. The tertiary structure of ubiquitin has well separatedtermini, allowing for attachment of one or more nucleotides at oneterminus, and one or more dyes at the other terminus to provideseparation and shielding. For example, mutation of the native lysines toarginines results in a unique reactive amine at the N-terminus, andaddition of a cysteine residue near the C-terminus provides a uniquereactive thiol. See, e.g. Vijay-Kumar, S., Bugg, C. E., Cook, W. J.,(1987) J. Mol. Biol. 194: 531-544 incorporated herein by reference inits entirety for all purposes. In some cases the ubiquitin will have ahis tag such as a hexa-his tag at its N or its C terminus.

FIG. 3 shows an image representing the three dimensional structure ofubiquitin 1UBQ. An arginine close to the C-terminus and the N-terminalmethionine are indicated as dotted spheres. The locations of the lysinesare shown as spheres. All other amino acids are shown as sticks, and thebackbone is represented as a ribbon.

A sequence for ubiquitin is provided in SEQ ID NO:1 below:

SEQ ID NO: 1 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

One useful construct to segregate dye and nucleotide involves mutatingall the lysines to arginines, and mutating the arginine at position 74to a cysteine (R74C). This would leave a unique primary amine at theN-terminal Met1, and a unique thiol at position 74. Table 1 shows someubiquitin mutants useful as protein shields.

TABLE 1 pET11a.His6co.Ubiquitin.copET11.His6co.ENLYFQS.Ubiquitin.K6R_K11R_K27R_K29R_K33R_K48R_K63R_R74C.copET11.His6co.ENLYFQSG.Ubiquitin.K6R_K11R_K27R_K29R_K33R_K48R_K63R_R74C.copET11a.His6co.Ubiquitin.K29R_K48C_K63R_77D.copET11a.His6co.Ubiquitin.K29C_K48C_K63C.copET11.His6co.Ubiquitin.K6E_K11R_K27R_K29R_K33R_K48R_K63R_R74C.copET11.His6co.Ubiquitin.K6R_K11R_K27R_K29R_K33R_R42E_K48R_K63R_R74C.copET11.His6co.Ubiquitin.K6E_K11R_K27R_K29R_K33R_R42E_K48R_K63R_R74C.copET11.His6co.Ubiquitin.K6R_K11R_K27R_K29R_K33R_K48R_K63R_R74C.copET11.His6co.Ubiquitin.R74C.co

The protein TOP7 can also be used as a protein shield or a portion of aprotein shield. Top7 is an artificial 93-residue protein, which wasdesigned to have a unique fold not found in nature. See Kuhlman et. al.,(2003, Nov. 21). “Design of a novel globular protein fold withatomic-level accuracy”. Science 302 (5649): 1364-1368, U.S. patentapplication Ser. No. 12/429,930, and U.S. Pat. No. 7,574,306, eachincorporated herein by reference in their entirety for all purposes.FIG. 4 shows a three dimensional representation of TOP7 protein. Lysinesare shown as spheres; the N- and C-terminal residues that are visible inthe structure are shown as dotted spheres. As described above, thevarious residues can be mutated to allow for specific attachment of oneor more dyes or one or more nucleotides to the protein.

A sequence for TOP7 is provided in SEQ ID NO:2 below:

SEQ ID NO: 2 MGDIQVQVNIDDNGKNFDYTYTVTTESELQKVLNELMDYIKKQGAKRVRISITARTKKEAEKFAAILIKVFAELGYNDINVTFDGDTVTVEGQLEGGSLE HHHHHH

Representative TOP7 mutants are shown below in Table 2.

TABLE 2 pET11a.TOP7.co.His6copET11a.TOP7.K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R_100.1C.co.His6copET11a.BtagV07co.TOP7.100.1C.co.His6copET11a.TOP7.K15R_K41R_K42R_K46R_K62R_K69R_110.1C.co.His6copET11a.TOP7.K15R_K46R_R49C.co.His6co pET11a.TOP7.100.1C.co.His6copET11.TOP7(1-100).AAAR.(EAAAR)8.TOP7_2(1-100).K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R_100.1C.His6copET11.TOP7(1-100).AAAR.(EAAAR)8.C.His6co pET11.TOP7(1-100).K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R.AAAR.(EAAAR)8.TOP7_2(1-100).K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R_100.1C.His6copET11.TOP7(1-100).K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R.AAAR.(EAAAR)8.C.His6copET11.TOP7(1-100).PKSERRS(32-100).K35R_K43R_K45R_K56R_K63R_K66R_K67C_K83R_K96R_K97R_K98R.His6copET11.TOP7(1-100).K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R.PKSERRS(32-100).K35R_K43R_K45R_K56R_K63R_K66R_K67C_K83R_K96R_K97R_K98R.His6copET11.TOP7(1-13).GG.PKSERRS(32-100).-K35R_K43R_K45R_K56R_K63R_K66R_K67C_K83R_K96R_K97R_K98R.-K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R.GG.TOP7(14-104).His6co

Coiled-coils of alpha helices, or single, stable alpha helices, provideparticularly efficient means by which to generate two widely separatedpoints of attachment in a protein scaffold. An example of a coiled-coilis provided by thremostable Seryl tRNA synthetase from Pyrococcushorikoshii (PKSERRS) as shown in its crystal structures (for example,Protein Data B ank database ID 2ZR2). The coiled-coil domain of SeryltRNA synthetases can be transplanted into different protein scaffolds,as shown by the crystal structure of dynein-SeryltRNA synthetase(Protein Data Bank ID 3ERR). A stable helix in solution can be generatedby using a repeat of the sequence EAAAR (Huyghes-Despointes, et al.1993).

Particularly useful proteins protein shields for the invention includeavidin protein including avidin, streptavidin, tamavidin, traptavidin,xenavidin, bradavidin, AVR2, AVR4, and homologs thereof. In some casesthe monomeric, dimeric, or tetrameric forms can be used. In particular,the tetrameric form of the avidin protein in combination with bis-biotinlinked dye components and/or nucleotide components are useful in proteinshielded nucleotide analogs. In some cases, glycosylation variants ofthe avidin proteins are used.

The protein shield of the invention can be based on or include theprotein tamavidin and its homologs. Tamavidin is a fungal avidin-likeprotein that binds biotin with high affinity. FIG. 5 shows a crystalstructure of the tetrameric faint of tamavidin, with two subunits shownin dark, and two in light; gray. The biotins are shown in sphererepresentation. See e.g. RCSB Protein Data Bank protein code 2ZSC andTakakura, et al., Journal: (2009) 276: 1383-1397, incorporated herein byreference in its entirety. Tamavidin may be mutated for example the C135can be mutated in case the cysteine would have some unwanted reactivity.In some cases tamavidin will be constructed to have his tag at its N orC terminus. Tamavidin can be advantageous in that it can be more stablethan streptavidin and can be more soluble in E. coli expression.

Sequences of the monomeric protein that makes up the tetramerictamavidin proteins (Tam1 SEQ ID NO:3 and Tam2 SEQ ID NO:4) are listedbelow.

SEQ ID NO: 3 MKDVQSLLTGTWYNELGSTMNLTANKDGSLTGTYHSNVGEVPPTYHLSGRYNLQPPSGQGVTLGWAVSFENTSANVHSVSTWSGQYFSEPAEVILTQWLLSRSSEREDLWQSTHVGHDEFSKTKPTKEKIAQAQLLRRGLKFE SEQ ID NO: 4MSDVQSSLTGTWYNELNSKMELTANKDGTLTGKYLSKVGDVYVPYPLSGRYNLQPPAGQGVALGWAVSWENSKIHSATTWSGQFFSESSPVILTQWLLSSSTARGDVWESTLVGNDSFTKTAPTEQQIAHAQLHCRAPRLK

One useful particularly avidin protein is streptavidin, and inparticular in the tetrameric form. A sequence of the monomer thatassociates to form the tetrameric form of streptavidin is provided inSEQ ID NO:5 below:

SEQ ID NO: 5 MEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS

Papain also provides a protein structure for use as a protein shield oras part of a protein shield. Papain, also known as papaya proteinase I,is a cysteine protease enzyme present in papaya. Proteins in the papainfamily, which are present in many species can also be used as proteinshields. FIG. 6 shows a representation of papain indicating the positionof lysines and of a reactive cysteine in the active site. As describedabove, amino acid sites can be mutated to provide the appropriate sitesfor attachment of dye and nucleotide components.

Another suitable protein for use as a protein shield is maltose bindingprotein. Maltose binding protein is a part of the maltose/maltodextrinsystem of Escherichia coli, which is responsible for the uptake andefficient catabolism of maltodextrins. Maltose binding protein has anapproximate molecular mass of 42.5 kilodaltons. FIG. 7 shows a threedimensional representation of maltose binding protein with lysines shownin stick figures. Wild type maltose binding protein has no cysteines;some residues that have been mutated in the literature to generatecysteines are shown as spheres. Table 3 shows some suitable constructsfor maltose binding protein. These constructs without the his-tag canalso be useful. These constructs contain multiple naturally occurringlysines along with literature based unique engineered cysteines.

TABLE 3 pET11.His10co.MBP.co pET11.His10co.MBP.S337C.copET11.His10co.MBP.N100C.co pET11.His10co.MBP.S233C.co

Bamase/bamstar proteins can also be used as protein shields in theinvention. Barnase is a bacterial protein that has about 110 amino acidsand has ribonuclease activity. It is synthesized and secreted by thebacterium Bacillus amyloliquefaciens, and is lethal to the cell whenexpressed without its inhibitor barstar. Barstar binds to and occludesthe ribonuclease active site, preventing barnase from damaging thecell's RNA after it has been synthesized but before it has beensecreted. The barnase/barstar complex has an extraordinarily tightprotein-protein binding. Either barnase, barstar, or the barnase/barstarcomplex can be used as a protein shield. In some embodiments, one ormore nucleotides are attached to barnase, and one or more dyes areattached to barstar, then the proteins are combined to form the barnasebarnstar with the nucleotides and dye substituents separated such thatcontact between the dye and the polymerase associated with thenucleotide component does not occur. The opposite approach with one ormore dyes on barnase and one or more nucleotides on barstar can also beused. FIG. 8 shows a three dimensional representation of thebarnase/barstar complex. Barstar is shown as a ribbon with lysinesindicated as spheres.

SNAP-tag protein can be used as a shield protein in the invention.SNAP-tag is a 20 kDa mutant of the DNA repair proteinO6-alkylguanine-DNA alkyltransferase that reacts specifically andrapidly with benzylguanine (BG) derivatives, leading to irreversiblelabeling of the SNAP-tag with a synthetic probe. SNAP-tag protein hasabout 184 residues. In some cases one or more nucleotides are attachedto the SNAP tag protein, and one or more dyes are attached to abenzylguanine derivate. In some cases, each of these can be madeseparately, then combined to form the nucleotide analog having theshielding protein. A similar approach can be take in which the one ormore dyes are attached to the SNAP-tag, and the one or more nucleotidesare attached to the benzylguanine derivative. FIG. 9 shows a threedimensional version of a SNAP tag protein associated with benzylguaninehaving lysines shown as spheres. See, for example, RCSB Protein DataBank code 3KZZ.

Another type of protein that can be a protein shield or componentthereof is a beta lactamase. Beta lactamases are enzymes produced bysome bacteria that confer resistance to beta-lactam antibiotics. Thebeta lactamases react by opening up the beta lactam ring in theantibiotic. In some embodiments, a beta lactamase suicide inhibitor isused to connect one or more dye components or one or more nucleotidecomponents to the beta lactamase, where the beta lactamase carries theother component. For example, the beta lactamase inhibitor clavulanicacid attached to one or more dyes can be reacted with a beta lactamaseattached to one or more nucleotides. The clavulanic acid forms acovalent bond with the enzyme attaching the nucleotide components. Theattachments to the protein are arranged in order to prevent contactbetween the dyes and a polymerase enzyme associated with one of thenucleotide substituents. FIG. 10 shows a three dimensionalrepresentation of a beta lactamase enzyme associated with an inhibitorattached to a fluorescein label. Suitable beta lactamases includecephalosporinases, penicillinases, carbenicillinases, andcarbapenamases.

The coiled-coil domain of a serine tRNA synthetase can be used as aprotein shield or component of a protein shield. This domain has a rigidstructure that can provide separation between the dye and nucleotidecomponents. FIG. 11 shows a three dimensional representation of a coiledcoil domain of a serine tRNA synthetase. The coil structure can be fusedto the terminus of a single domain protein. A mutation such as acysteine can be incorporated into the tip of the coiled coil domain,which can be attached to one or more nucleotides. One or more dyes areattached to more distant portion of the coil coiled domain or to theprotein to which the domain is fused. Alternatively, the one or morenucleotides can be attached to the mutation at the tip of the domain,and the one or more dyes can be attached to a more distant portion ofthe domain or to the protein to which it is fused.

Other suitable shield proteins include proteins engineered to includeLeucine Rich Repeats such as Ankyrin repeats, Cyanoverin, and Protein G.

One suitable approach which is embodied in some of the examples providedis the use of tandem domains—protein domains that are associated, one ofwhich has the nucleotide moieties, and one of which has the dyemoieties. In some cases, the tandem domains can have an affinity for oneanother, in some cases the tandem domains can be connected usingcovalent or binding pair chemistry, in other cases, the tandem domainscan be fused and expressed together in cloning. Tandem domains that areconnected subsequent to attachment of the nucleotide and dye moieties,e.g. by affinity, covalent or binding pair chemistry are useful becausethis allows for selective chemistry for attachment of each of the typesof moieties. It also allows for a cassette type approach where differentnucleotide types can be combined with different dye types in arelatively simple synthetic scheme.

Chemical linking of domains can be carried out in a variety of waysincluding the chemical methods described herein. One example is to use aTOP7 construct with a unique cysteine thiol. Two batches of such aconstruct are prepared: one in which the thiol is labeled with aterminal alkyne (using, e.g. a maleimide linked alkyne); the other whereit is labeled with an azide. The amines in one batch can then be labeledwith bases, and the other with dyes. Subsequently, the specificity of aClick reaction can be exploited to generate covalently linkedheterodimers. The linked proteins can be the same or a differentprotein. As will be clear to one of skill in the art similar schemes canbe applied to proteins or protein domains including those describedherein including for ubiquitin, or ubiquitin/top7, etc.

One approach is to use a his-tagged protein as one of the elements ofthe pair, and maltose binding protein as the other element. A nickelcolumn (selective for binding poly-histidine) followed by an amylosecolumn (which would retain maltose binding protein) results in thepurification of heterodimers from any contaminating homodimericfractions.

Pairs of tightly binding proteins that can be used for the production oftandem-protein protein shields are well known in the art. There are manystrong protein-protein interactions including many protein-proteininhibitor interactions. Suitable systems include barnase/barstar,colicin immunity proteins, leucine rich repeat containing proteins,ribonuclease inhibitors, or coiled-coil proteins.

The connection of proteins to produce tandem-protein protein shields canbe done with small molecules. For example, dihydrofolate reductase(DHFR) binds to the drug methotrexate. Crabtree et al. have demonstratedthat a dimeric methotrexate can induce dimerization of DHFR. Thisapproach can be used to generate heterodimers of base and dye labeledDHFR.

The tandem domains can be formed by fusing of the domains with cloning.Suitable fused tandem domains include TOP7-TOP7, TOP7-Ubiquitin, orcoiled-coil fused to TOP7 or ubiquitin.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a polymerase, or the aminoacid sequence of a polymerase) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90%, about95%, about 98%, about 99% or more nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Such “substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

Proteins and/or protein sequences are “homologous” when they arederived, naturally or artificially, from a common ancestral protein orprotein sequence. Similarly, nucleic acids and/or nucleic acid sequencesare homologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence similarity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofsimilarity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence similarity over 50, 100, 150 or more residues is routinely usedto establish homology. Higher levels of sequence similarity, e.g., 30%,40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% or moreidentity, can also be used to establish homology. Methods fordetermining sequence similarity percentages (e.g., BLASTP and BLASTNusing default parameters) are described herein and are generallyavailable.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g, bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyCurrent Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., supplemented through 2012).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Nucleotide Analogs Comprising Avidin Proteins and Bis-Biotin Linkages

Avidin proteins are useful proteins in nucleotide analogs of theinvention. Avidin proteins are biotin-binding proteins, generally havingfour biotin binding sites. Avidin proteins include, for example, avidin,streptavidin, tamavidin, traptavidin, xenavidin, bradavidin, AVR2, AVR4,and homologs thereof. The biotin binding sites provide attachment pointsfor moieties having one or more nucleotides or one or more fluorescentdyes. Unless otherwise specified, the term avidin protein as used in theapplication refers to the tetrameric form of the protein. In some casesglycosylation variants are used. We have found that compounds having twobiotin sites attached to the tetrameric protein are very useful forproducing nucleotide analogs in which the nucleotide portion is keptaway from the dye portion such that the dye is prevented from directlyinteracting with the enzyme.

FIG. 14 illustrates the way that bis-biotin compounds can be used toproduce protein-shield nucleotide analogs. In FIG. 14(A) a bis-biotinmoiety is attached to a dye component. The bis-biotin is attached to twoof the four streptavidin biding sites such that the dye component isheld onto one side of the avidin protein. In FIG. 14(B) the nucleotidecomponent is attached through the bis-biotin linkage. In FIG. 14(C),both the dye component and the nucleotide component are attached to thestreptavidin via a bis-biotin moiety. Although the avidin protein-biotinbond is very strong, we have found that with a single attachment site,there can be some exchange over time. While in many applications, asmall amount of exchange is not problematic, we have found that wherethese connections are used to produce nucleotide analogs for singlemolecule sequencing, such an exchange can cause a degradation inperformance. Where the nucleotide analog is prepared using bis-biotinlinkages, we have found that the shelf-life of the nucleotide analogs issignificantly improved. The compounds of 14(C) thus provide an improvedability to separate the dye component from the enzyme in order toimprove the photostability of the sequencing system, and these compoundsalso show unexpected improvements in shelf-life.

For the compounds shown in FIGS. 14(A) and 14(B), typically, thecompounds are made by first reacting the bis-biotin moiety with theavidin protein. With the appropriate bis-biotin moiety, the reaction ofthe second biotin is rapid, as the first biotin reaction holds thesecond biotin in proximity the reaction site on that avidin protein.Once the bis-biotin portion is reacted with the avidin protein, thecompound can be readily separated from other reaction componentsincluding unreacted avidin protein. This compound can then be reactedwith the single biotin component to form the structure shown in FIGS.14(A) and 14(B). Typically the identity of the single-biotin component(the nucleotide component in 14(A) and the dye component in 14(B) is thesame. In some cases, different components can be present in each of theother two binding sites on the avidin protein. This can be accomplished,for example, with a stepwise process including an intermediatepurification of the compound in which only one of the two remainingsites have reacted.

In the case of 14(C) the preparation of the compound can proceed byadding either of the bis-biotin moieties to the avidin protein in afirst step followed by a second step of adding the nucleotide component.This can be done with or without an intermediate purification step. Thefinished product 14(C) can be readily purified from other componentsincluding unreacted starting materials, e.g. by chromatography.

The dye component can have one or more dye moiety. For example, thenucleotide analog can have from about 1 to about 100 dye moieties, about1 to 50 dye moieties, about 1 to about 18 dyes moieties, or 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 dye moieties. Insome cases, the nucleotide analog has at least about 1 to about 18 dyesmoieties, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, or 18 dye moieties. In some cases the dye componentincludes FRET dyes, for example having one donor and one acceptor, twodonors and one acceptor, two donors and two acceptors, etc. The numberof dyes can be selected and readily tested for performance. In general,having more than one dye can be used to obtain higher brightness, but asis known in the art, the addition of one more dye does not alwaysincrease the brightness commensurate with the number of dyes. Those ofskill in the art will understand how to attach the dyes and chose thenumber of dyes with the best performance for a given system. The type oflinkers used to attach the dyes including the length of the linker andits chemical functionality can also be used to engineer the appropriatelabel performance. Typically the dye moieties are fluorescent dyes. Insome cases, the dye moieties comprise fluorescent particles, or otherluminescent species.

The nucleotide component can have one or more phospholinked nucleotide.In some cases, the invention may refer to a nucleotide, and in othercases to a nucleoside. Typically a nucleoside has no phosphates where anucleotide has at least one phosphate linkage. Thus a nucleosidephosphate may be seen as a nucleotide as it has at least one phosphate.Those of ordinary skill in the art will understand the meanings of theterms as used herein by the context. It is important for many real-timesingle molecule systems that the nucleotide moiety be phospholinked. Inthis way, the cleavage of the alpha-beta phosphodiester bond in thenucleotide analog releases the labeled component. For example, thenucleotide analog can have from about 1 to about 100 phospholinkednucleotide moieties, about 1 to 50 phospholinked nucleotide moieties,about 1 to about 18 phospholinked nucleotide moieties, or 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phospholinkednucleotide moieties. In some cases, the nucleotide analog has at leastabout 1 to about 18 phospholinked nucleotide moieties, or at least about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18phospholinked nucleotide moieties. Raising the number of phospholinkednucleotide moieties tends to raise the effective concentration of thenucleotide at the enzyme. As is well known in the art, the concentrationof nucleotide can be varied in order to control the polymerase kinetics,and that depending on the system and the desired performance, theconcentration of the nucleotide can be varied both by controlling theamount of nucleotide analog per volume and by controlling the number ofphospholinked nucleotides per nucleotide analog. Those of skill in theart will understand how to use the compounds of the invention tooptimize system performance. The lists of potential choices describedherein for the numbers and types of phospholinked moieties can becombined with any of the described numbers and types of dye moietiesdescribed.

FIG. 15 shows an exemplary compound of the type shown in 14(A) in whicha dye moiety is attached to an avidin protein, such as streptavidin,through a bis-biotin moiety, and nucleotide moieties are attached to theavidin protein through single biotin binding moieties. The figure showsa representative bis-biotin dye having a central tri-functional linkerT1 connecting one dye moiety to two biotins. The trifunctional linker isconnected to the biotin moieties through spacers or linkers designatedby an X. A typical spacer or linker could have, for example three to sixmethylene units (CH₂) connected through amide linkages. The bis-biotinwith the dye component is attached to two of the streptavidin bindingsites. The other two streptavidin binding sites are attached, each to asingle biotin of a biotin dinucleotide that is made up of atrifunctional linker T7 attached to the biotin through a linker orspacer, and to two phospholinked nucleotides. Here, the phospholinkednucleotides are dT6P, a deoxy T nucleoside connected to the linkerthrough a hexaphosphate. We have found that relatively rigid, aromatictrifunctional linkers such as T1 and T7 can be useful in theconstruction of the nucleotide analogs of the invention. The figureshows a representative structure to illustrate how such a nucleotideanalog of the invention is made.

FIG. 16 shows two representative bis-biotin linkers that can be used inthe instant invention. We have found that the length of both of thesebis-biotin linkers is useful for the invention. The linker shown in FIG.16(A) has 40 bonds between the biotin linkages. The linker shown in FIG.16(B) has 26 bonds between the biotin linkages. As will be understood,if the linkage between the biotins is too short, then the two biotinswill not readily connect to two binding sites on the same avidintetrameric protein. Also, if the linkage between the biotins is toolong, then the second biotin may be slower to contact and bind to thesecond binding site on the avidin protein. In some cases, a bis-biotinlinker having between about 15 and about 50 bonds between biotinlinkages is useful. The bis-biotin linkers of FIG. 16 are shown aslinked to a component. This component can be either dye component or anucleotide component, and as consistent with the application, the dyecomponent can have one or multiple dye moieties, and the nucleotidecomponent can have one or multiple phospholinked nucleotide moieties.

FIG. 17 shows a nucleotide analog of the invention having two dyemoieties attached to an avidin protein shield through a bis-biotinmoiety, and having four phospholinked nucleotide moieties. FIG. 17 showsthe structure of a bis-biotin double dye compound that can be used toproduce such a nucleotide analog. Here, the bis-biotin double-dye analogis produced using two trifunctional linkers T2 and T1. Each of the twobiotins is attached to the trifunctional linker through two aminohexanamide spacers X through amide linkages. This structure illustratesone approach that can be expanded to incorporate even more dye moietiesor phospholinked nucleotide moieties. In the nucleotide analog of FIG.17, the two dyes can be dyes of the same type, or can be of twodifferent types. The two dyes can comprise a FRET donor and a FRETacceptor. FRET pairs can be used, for example in order to produce labelsthat are bright, and that have a relatively high Stokes shift, where theStokes shift is the difference in wavelength between the absorptionmaximum and emission maximum for the label. Here, two phospholinkednucleotides are connected to a trifunctional linker, T7, which isconnected through a linker or spacer X to a single biotin. As describedabove the preparation of the nucleotide analog typically involves afirst reaction between the avidin protein such as streptavidin and thebis-biotin double dye, followed by treatment with the compound with asingle biotin moiety and two phospholinked nucleotides.

FIG. 18 provides an example of a how a bis-biotin double dye compoundcan be formed using standard organic chemistry methods. The dye moietyfor the present synthesis 1800has a single activatable carboxylic acidwhich is coupled to the amine group of 6-amino-hexanoic acid. This acidgroup on the compound formed, 1801, is activated and reacted withtrifunctional linker T2 to form a double dye species 1802. Thecarboxylic acid on 1802 is activated and reacted with trifunctionallinker T1 to form compound 1803. Compound 1803 is reacted with theN-hydroxy succinimide ester biotin-X-NHS where X is a6-amino-hexanamide. This condensation forms the bis-biotin double dyecompound 1804.

FIG. 19 shows a nucleotide analog of the invention having 8phospholinked nucleotides. For this compound, there is a bis-biotinmoiety attached to a single dye moiety that is attached to two of thefour avidin protein, e.g. streptavidin, biotin binding sites. Thephospholinked nucleotides are connected through a compound having onebiotin and three trifunctional linkers (e.g. one T7 trifunctional linkerand two Sh trifunctional linkers). The phospholinked nucleotide moietiescan each be, for example a hexaphosphate connected to nucleoside dT(dT-6-P). FIG. 19 also shows the structure of Sh, a trifunctional linkerthat can be used to produce the nucleotide analog shown. For thecompound of interest, the amine group and two acetylene groups of the Share used to link the Sh, and the carboxylic acid group is leftunreacted. As discussed in other parts of the application, in some case,having a charged groups on the nucleotide analog such as carboxylate andsulfate groups can assist in assuring the aqueous solubility of thenucleotide analogs. The trifunctional linker Sh is only one possibletrifunctional linker that can be used in the instant application. Insome cases it is desirable to control the length of the set of bondsconnecting the linking groups. FIG. 19 also shows an Sh alternativewhere the lengths of these sets of bonds can be varied to improveperformance of the nucleotide analog.

FIG. 20 shows an example of a compound having a single biotin and fourphospholinked nucleotides that can be used to produce nucleotide analogsof the invention. Here the biotin is attached to a T7 trifunctionallinker through an amino hexanoic acid spacer. The trifunctional liner T7is connected to two Sh trifunctional linkers, and each of the Shtrifunctional linkers is attached to two phospholinked nucleotidemoieties through a linker. The phospholinked nucleotide moieties areattached to the Sh through triazole linkages that can be formed, forexample using Click chemistry.

FIG. 21 shows a nucleotide analog of the invention of the inventionhaving 12 phospholinked nucleotide moieties. For this compound, there isa bis-biotin moiety attached to a single dye moiety that is attached totwo of the four avidin protein, e.g. streptavidin, biotin binding sites.In some cases, this bis-biotin portion can have multiple dye moieties asdescribed herein. The phospholinked nucleotides are connected through acompound having one biotin, one trifunctional linker T7 and twotetra-functional linkers (e.g. Sh tetra-functional linkers). The Shlinker in this example differs from the Sh linker referred to above andshown in FIG. 19 and FIG. 20 in that it has an additional linkagethrough the carboxylic acid shown in the Sh structure on FIG. 19. Thephospholinked nucleotide moieties can each be, for example ahexaphosphate connected to nucleoside dT (dT-6-P).

FIG. 22 shows an example of a compound having a single biotin and sixphospholinked nucleotides that can be used to produce nucleotide analogsof the invention. Here the biotin is attached to a T7 trifunctionallinker through an amino hexanoic acid spacer. The trifunctional liner T7is connected to two Sh tetra-functional linkers, and each of the Shtetrafunctional linkers is attached to three phospholinked nucleotidemoieties through a spacer. The phospholinked nucleotide moieties areattached to the Sh through triazole linkages that can be formed, forexample using Click chemistry.

FIG. 23 shows a structure for a nucleotide analog of the invention inwhich both the phospholinked nucleotide moieties and the dye moietiesare attached to the avidin protein through bis-biotin moieties. Here,two dye moieties are attached to a bis-biotin moiety through atrifunctional linker T2. The biotin moieties are connected through aspacer X and a trifunctional moiety T1. There are 6 phospholinkednucleotide moieties attached to the bis-biotin moiety in two groups ofthree phospholinked nucleotides each attached through a tetrafunctionallinker G to a trifunctional linker. FIG. 23 illustrates that thenucleotide analog of the invention can be prepared by reacting an avidinprotein such as a streptavidin with a bis-biotin dye component and abis-biotin hexanucleotide component. These reactions are typicallycarried out in series, optionally with a purification step between thereactions. This figure shows an analog with two dye moieties and sixphospholinked nucleotide moieties, but it will be clear to those ofordinary skill in the art that the methods provided allow for thepreparation of nucleotide analogs with a wide variety of numbers andtypes of substituents.

FIG. 24 shows an example of a bis-biotin hexanucleotide compound forpreparing a nucleotide analog of the invention. This nucleotide analoghas two biotins, each connected through an amino hexanamide spacer to atrifunctional linker T. The trifunctional linker T is connected to a 1,3, 5-triamino cyclohexane linker, which is in turn connected to twotetrafunctional G linkers which are each connected to threephospholinked nucleotide moieties. The phospholinked nucleotide moietiesare attached to the tetrafunctional linkers G through triazole linkagesthat can be formed, for example using Click chemistry.

FIG. 25 shows components that are used in the preparation of thebis-biotin hexanucleotide shown in FIG. 24 using standard organicchemical techniques.

FIG. 26 shows a representative synthesis of a bis-biotin hexanucleotidefor preparing a nucleotide analog of the invention. Two equivalents ofBoc-protected 4-aminopiperidine is added to 2, 4,6-trichloro-1,3,5-triazine to form intermediate 2601, to which is added1,3,5-triaminocyclohexane to form intermediate 2602. To intermediate2602 is added two equivalents of the substituted gallic acid 2603 formamide bonds using eitherBenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP) orO-(7-azabenzotriazole-1-yl)-N,N,N,N′-tetramethyluroniumhexafluorophosphate (HATU), producing intermediate 2604. Removal of theBac protecting groups with acid yields intermediate 2605 which isreacted with two equivalents of biotin-X-NHS (the N-hydroxysuccinimideester of biotin-X shown in FIG. 25) to yield intermediate 2606. Clickchemistry (Copper catalysed Huisgen addition) is then used to attach sixequivalents of phospholinked nucleotide intermediate 2607 to form thebis-biotin hexanucleotide shown in FIG. 24.

FIG. 27(A) shows a protein shield nucleotide analog of the inventioncomprising two avidin proteins. The analogs have a dye component that ispart of a central tetra-biotin compound having two bis-biotin moietiesand having two nucleotide components, each attached to one of the avidinproteins through a bis-biotin moiety. The dye components, nucleotidecomponents, bis-biotin moieties, and avidin proteins can comprise any ofthose referred to or described herein. The dye component can compriseany suitable number and type of dye moiety, and the nucleotide componentcan comprise any suitable number and type of phospholinked nucleotidemoiety. As described herein, these types of protein shielded nucleotidescan be made to prevent the dye component from directly interacting withthe polymerase, thereby improving the photostability of a real-timesingle molecule sequencing system. The nucleotide components attached toeach of the two avidin proteins can be the same or different components.In some cases, the two nucleotide components are the same.

The dye component can have one or more dye moiety. For example, thenucleotide analog can have from about 1 to about 100 dye moieties, about1 to 50 dye moieties, about 1 to about 18 dyes moieties, or 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 dye moieties. Insome cases, the nucleotide analog has at least about 1 to about 18 dyesmoieties, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, or 18 dye moieties. In some cases the dye componentincludes FRET dyes, for example having one donor and one acceptor, twodonors and one acceptor, two donors and two acceptors, etc. The numberof dyes can be selected and readily tested for performance. In general,having more than one dye can be used to obtain higher brightness, but asis known in the art, the addition of one more dye does not alwaysincrease the brightness commensurate with the number of dyes. Those ofskill in the art will understand how to attach the dyes and chose thenumber of dyes with the best performance for a given system. The type oflinkers used to attach the dyes including the length of the linker andits chemical functionality can also be used to engineer the appropriatelabel performance. Typically the dye moieties are fluorescent dyes. Insome cases, the dye moieties comprise fluorescent particles, or otherluminescent species.

The nucleotide component can have one or more phospholinked nucleotide.It is important for many real-time single molecule systems that thenucleotide moiety be phospholinked. In this way, the cleavage of thealpha-beta phosphodiester bond in the nucleotide analog releases thelabeled component. For example, the nucleotide analog can have fromabout 1 to about 100 phospholinked nucleotide moieties, about 1 to 50phospholinked nucleotide moieties, about 1 to about 18 phospholinkednucleotide moieties, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, or 18 phospholinked nucleotide moieties. In some cases, thenucleotide analog has at least about 1 to about 18 phospholinkednucleotide moieties, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, or 18 phospholinked nucleotide moieties.

Typically, the nucleotide analogs of FIG. 27(A) are formed by a firstreaction of the dye component having two bis-biotin moieties to form anintermediate in which each of the two avidin proteins (e.g.streptavidin) have two open binding sites. This reaction can be carriedout, for example, by using an excess of avidin protein, then purifyingthe desired compound from the unreacted avidin protein. The nucleotidecomponents comprising bis-biotin moieties are then connected to form thenucleotide analog. Where the two nucleotide analogs are the same, thiscan be accomplished in one step, typically followed by purification.

While it is typically preferred to have the dye component between thetwo avidin proteins, and have the nucleotide components on the outsideof the two avidin proteins, in some cases the reverse configuration isused where then nucleotide component is connected to the centraltetra-biotin comprising two bis-biotins, and dye components are on theoutside of the avidin proteins connected through bis-biotin moieties.For these constructs the dye components on each of the avidin proteinscan be the same or can be different.

FIG. 27(B) shows an exemplary nucleotide analog of the inventioncomprising two avidin proteins. The two avidin proteins, e.g.streptavidin, are connected with a tetra-biotin compound having twobis-biotin moieties connected to one another through a trifunctionallinker Sh. The trifunctional linker Sh is also connected totrifunctional linker T2 which has two dye moieties connected to it inwhich one dye moiety is a FRET donor connected through a linker X, andthe other dye moiety is a FRET acceptor. The nucleotide analog has 12phospholinked nucleotides with 6 phospholinked nucleotides attached toeach avidin protein. The phospholinked nucleotides are for example dGconnected through a hexaphosphate (dG6P). These are connected in groupsof three to a tetrafunctional linker G, which is connected to thebis-biotin moiety through a trifunctional linker

FIG. 27(B) also illustrates that the nucleotide analog having two avidinproteins can be formed from streptavidin, a tetra-biotin having a dyecomponent, and a bis-biotin hexanucleotide. Typically the preparationwould be carried out in two steps, first coupling the tetra-biotin dye,then subsequently coupling the bis-biotin hexanucleotide.

FIG. 28 shows an example of a tetra-biotin dye component that can beused to produce nucleotide analogs of the invention. The two bis-biotinmoieties have biotins connected to a trifunctional linker T8 through anamino hexanamide linker. These bis-biotin moieties are each connected toa trifunctional linker Sh, which is also connected to a trifunctionallinker T2 which has donor and acceptor dyes connected to it. Thistetra-biotin dye component can be prepared using standard organicchemistry.

We have found that it can be quite advantageous for the performance ofthe protein shielded nucleotide analog to have multiple charged groups.In some cases the multiple charged groups are anionic. In some cases themultiple charged groups comprise carboxylate, sulfonate, sulfate orphosphate groups. In one preferred approach, the protein shieldnucleotide analog comprises multiple sulfonate (—SO₃ ⁻) groups. Forexample, the nucleotide analog can have 6 to 50 sulfonate groups, 9 to40 sulfonate groups, or 10 to 30 sulfonate groups. In some cases,multiple sulfonate groups are included in a bis-biotin compoundcomprising the nucleotide component. In some cases, the bis-biotincomprising the nucleotide component has from 6 to 50 sulfonate groups, 9to 40 sulfonate groups, or 10 to 30 sulfonate groups. In some cases thebis-biotin comprising the nucleotide component has 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 sulfonate groups. In some cases, the his-biotin comprising thenucleotide component at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 sulfonategroups.

We have found that one particularly useful way to introduce sulfonategroups into the nucleotide analog is to include one or more six memberedaromatic rings each having multiple sulfonate groups attached to it,which we refer to as a kinetic modifier group, for example, a sixmembered aromatic ring having 2, 3, 4, or 5 sulfonate groups attached.One particularly useful group for attaching multiple sulfonate groups tothe nucleotide analogs of the invention is an SG group as shown below:

where m, n, and p are independently selected 1-18. In some cases m, n,and p are each 3, 4, 5, or 6. In some cases m, n, and p are each 3.

We have found that the inclusion of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 of these kinetic modifier groups can produce a protein shieldednucleotide analog with improved kinetic performance in sequencing.

FIG. 36 shows an exemplary bis-biotin compound having six phospholinkednucleotides and having four kinetic modifier groups. For this compoundfour kinetic modifier groups SG1, shown in the figure is used.

The structures below provides an exemplary bis-biotin compound havingtwo dye moieties for use in producing a nucleotide analog of theinvention.

where a=0-1; b=0-1; c=1-2; Dye 1 and Dye2 are selected from the Table 4.

The structure below provides an exemplary bis-biotin compound having asingle dye moiety.

where a 0-1; c=1-2; and Dye3 is selected from the Table 4.

TABLE 4 Exemplary Fluorescent Dyes

The exemplary compounds shown above are not meant to be limiting but toillustrate some ways in which one of ordinary skill can carry out theinvention. The compounds can be prepared by standard organic chemistrytechniques, and bis biotin compounds having any suitable type and numberof dye moiety can be analogously provided. As described herein, in somecases, the dye moieties comprise fluorescent dyes. Table 4 providesstructures of some exemplary fluorescent dyes that can be used. Asdescribed herein, it is typically desired that the nucleotide analogs ofthe invention be used in aqueous solution, and therefore that the dyemoieties are soluble in water. As illustrated in the table, the dyemoieties of the invention will typically have polar and/or ionic groupsin order to provide solubility. A particularly useful solubilizing groupis a sulfonate (—SO₃ ⁻) group.

In some, the four binding sites on streptavidin can be thought of as twopairs of sites where the sites within the pair are closer together thanthe sites pairs are from one another. Thus, one can selectivelyfunctionalize one pair of sites with dye moieties, and the other pair ofsites with nucleotide moieties to ensure that they are far enough apartsuch that when the nucleotide moiety is associated with the polymeraseenzyme, the dye moiety does not come into contact with the polymeraseenzyme.

The protein shields of the invention do not typically include shortproteins and peptides of fewer than about 60 amino acids. Even wherethese oligo-proteins are rigid, such as poly-proline linkers in somecases are not included in the invention.

It is understood in the literature how to direct dyes and nucleotides todistinct monomers within the streptavidin tetramer. Protocols aredescribed for forming a tetramer containing 1, 2, 3, or 4 monomersincapable of binding biotin. See e.g. Howarth et al Nature Methods 2006,which is incorporated herein by reference for all purposes. Theintroduction of single cysteine residues within streptavidin can also beused for example for providing conjugation sites that are chemicallyorthogonal to biotin. In one example of a heterotetramer a mixedheterotetramer with 2 subunits containing reactive cysteines, and twosubunits capable of binding biotin is used. In another example, thesubunits can be assembled separately. A maleimide PEG-N3 and maleimidePEG alkyne can be assembled using click chemistry. This yields anassembly with a large spatial segregation of dye and nucleotide. Anysuitable dye (or nucleotide) conjugated with a single reactive PEGmoiety may also be substituted for one of the tetramers.

Any of the nucleotide moieties or fluorescent moieties can be connectedto the shielding protein by a linker. The linker can have any suitablemolecular structure. It can include, for example, alkanes, hydroxyls,phosphates, peptides, glycols, or saccharide linkages. It is generallypreferred that a polar or hydrophilic linker be used in order to enhancewater solubility. The length of the linker is selected in order to allowthe moiety freedom to move with respect to the protein, but to preventcontact of the fluorescent moiety with the polymerase when thenucleotide moiety is associated with the polymerase.

Polar and ionic groups are also often added to portions of thenucleotide analog in order to improve water solubility as mostsequencing reactions are carried out in aqueous environments. Forexample, carboxylic acid groups, sulfate groups, sulfonate groups,phosphate groups and/or amine groups are added to the dye moieties,bis-biotin moieties, phospholinked nucleotide moieties or other portionsof the nucleotide analog to ensure adequate aqueous solubility. Inpreferred embodiments, as described herein, multiple sulfonate (—SO₃ ⁻)groups are attached to the linkers, in particular the linkers connectingthe phospholinked nucleotide moieties. We have found that the inclusionof the sulfonate groups can enhance the kinetic performance of thenucleotide analogs. One particularly useful way to introduce sulfonategroups into the nucleotide analog is to include one or more six memberedaromatic rings each having multiple sulfonate groups attached to it,which we refer to as a kinetic modifier group, for example, a sixmembered aromatic ring having 2, 3, 4, or 5 sulfonate groups attached.

In some cases, the rigidity of the linker is controlled in order to holdthe relevant component in the appropriate position. For example, rigidcomponents such as connected aromatic rings can be used in order tocontrol the rigidity of the linker. Another way to control the rigidityof the linker and the position of a dye or nucleotide component is touse a nucleic acid linker such as DNA or a derivative thereof such asPNA. For example, it is known that stretches of double stranded DNA canbe relatively rigid, allowing for controlling the position of thecomponent attached thereto. In some embodiments, the linkers comprisedouble-stranded nucleic acid portions such as double-stranded DNAportions.

The dye moieties can comprise any suitable luminescent label, Typicallythe dye moieties are fluorescent moieties. Fluorescent moieties can haveany suitable fluorescent dye or fluorescent particle or combinationthereof. The fluorescent moiety provides a signaling function, absorbingthe incident excitation light and giving off emitted light. Detectorsare used to determine the level of emitted light, allowing fordetermining whether a molecule having a given fluorescent moiety iswithin an observation volume. In sequencing reactions, generallymultiple nucleotide analogs are employed, each corresponding to adifferent base, and each emitting at a color that is distinct from theother analogs. In some cases, the dye moieties can comprisephosphorescent moieties.

A fluorescent moiety or fluorophore (F) can be selected from fluorescentlabeling groups including individual fluorophores and cooperativefluorophores, e.g., one or both members of a donor-quencher or FRETpair. In the case where F is at least one member of a cooperativefluorophore pair, the second member of the pair may also be includedwithin the F group, e.g., as a unified FRET dye structure (See, e.g.,U.S. Pat. No. 5,688,648 for a discussion of FRET dyes), or it may beprovided elsewhere on the analog or the overall system. For example, insome cases, the other member of the pair may be coupled to and as aportion of the Base moiety attached to the sugar group (See, e.g., U.S.Pat. No. 6,232,075 previously incorporated herein by reference).Alternatively, the other member of the pair may be coupled to anotherreaction component, e.g., a polymerase enzyme (See, e.g., U.S. Pat. No.7,056,676, previously incorporated herein by reference).

A wide variety of different types of fluorophores are readily availableand applicable to the compounds of the invention and includefluorescein, or rhodamine based dyes, cyanine dyes and the like. Avariety of such dyes are commercially available and include the Cy dyesavailable from GE Healthcare (Piscataway, N.J.), such as Cy3, Cy5, andthe like, or the Alexa® family of dyes available fromInvitrogen/Molecular Probes (Carlsbad, Calif.), such as Alexa 488, 500,514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, and 750.These fluorophores may be present as individual fluorophores or they maybe present in interactive pairs or groups, e.g., as fluorescent resonantenergy transfer (FRET) pairs.

Alternative labeling strategies may employ inorganic materials aslabeling moieties, such as fluorescent or luminescent nanoparticles,e.g. nanocrystals, i.e. Quantum Dots, that possess inherent fluorescentcapabilities due to their semiconductor make up and size in thenanoscale regime (See, e.g., U.S. Pat. Nos. 6,861,155, 6,699,723,7,235,361). Such nanocrystal materials are generally commerciallyavailable from, e.g., Invitrogen, Inc., (Calsbad Calif.). Again, suchcompounds may be present as individual labeling groups or as interactivegroups or pairs, e.g., with other inorganic nanocrystals or organicfluorophores.

Suitable fluorescent moieties are described in copending U.S. PatentApplications 2012/0077189, 2012/0058482, 2012/0058469, and 2012/0052506,which are incorporated herein by reference in their entirety for allpurposes.

In preferred aspects of the invention, the template nucleic acid is in acyclic form. Performing single-molecule sequencing on a cyclic nucleicacid template is advantageous in that it allows for redundant sequencingof a given region. The accuracy of a sequence determination can beimproved significantly by sequencing the same region multiple times.Cyclic nucleic acids that are highly useful for the current inventioninclude SMRT Bell™ templates, which are nucleic acids having a centraldouble-stranded region, and having hairpin regions at each end of thedouble-stranded region. The preparation and use of cyclic templates suchas SMRT Bells™, are described for example in U.S. patent applicationSer. No. 12/286,119, filed Sep. 26, 2008, and U.S. patent applicationSer. No. 12/383,855, filed Mar. 27, 2009, the full disclosure of whichis incorporated herein by reference for all purposes. One advantage ofthe SMRT Bell™ template is that it can be made from a library ofdouble-stranded nucleic acid, e.g. DNA, fragments. For example, a sampleof genomic DNA can be fragmented into a library of DNA fragments, byknown methods such as by shearing or by use of restriction enzymes. Thelibrary of DNA fragments can be ligated to hairpin adaptors at each endof the fragment to produce a library of SMRT Bell™ templates. Thehairpin adaptors provide single stranded regions within the hairpins. Byusing the same hairpin adaptor for all of the fragments, the hairpinadaptors, provide a position for universal priming of all of thesequences.

Methods for treating the surfaces of zero mode waveguides includingmethods for obtaining selective coupling to the base of the zero modewaveguides are described, for example, in U.S. Pat. Nos. 7,833,398,7,292,742 and in U.S. Patent Application Nos. 2008/0032301,2008/0241892, and 2008/0220537, the full disclosures of which areincorporated by reference herein for all purposes. In some cases, forexample biotin is selectively coupled to the base of the zero modewaveguide.

The template nucleic acid can be derived from any suitable natural orsynthetic source. In preferred embodiments, the template comprisesdouble stranded DNA, but in some circumstances double-stranded RNA orRNA-DNA heteroduplexes can be used. The template nucleic acid can begenomic DNA from eukaryotes, bacteria, or archaea. The template nucleicacid can be cDNA derived from any suitable source including messengerRNA. The template nucleic acid can be a library of double strandedsegments of DNA. The template nucleic acid can be linear or circular.For example, the nucleic acid can be topologically circular and have alinear double stranded region. A circular nucleic acid can be, forexample, a gapped plasmid. The nucleic acid is a double stranded linearDNA having a gap in one of the strands. The gap provides a site forattachment of the polymerase enzyme for nucleic acid synthesis. Thelinear double stranded DNA having a double-stranded DNA adaptor can bemade by ligation of DNA fragment to an adaptor through bluntend-ligation or sticky end ligation. The ligation produces a linear DNAhaving a gap close to the 5′ end of one or both of the strands. The gapcan be any suitable width. For example, the gap can be from 1 to 50bases, from 2 to 30 bases, or from 3 to 12 bases.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, nucleic acid analogs are included thatmay have alternate backbones, comprising, for example, phosphoramide,phosphorothioate, phosphorodithioate, and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506. The templatenucleic acid may also have other modifications, such as the inclusion ofheteroatoms, the attachment of labels, such as dyes, or substitutionwith functional groups which will still allow for base pairing and forrecognition by the enzyme.

The template sequence may be provided in any of a number of differentformat types depending upon the desired application. The template may beprovided as a circular or functionally circular construct that allowsredundant processing of the same nucleic acid sequence by the synthesiscomplex. Use of such circular constructs has been described in, e.g.,U.S. Pat. No. 7,315,019 and U.S. Patent Application No. 2009/0029385.Alternate functional circular constructs are also described in U.S.Patent Application 2009/0280538, and U.S. Patent Application2009/0298075, the full disclosures of each of which are incorporatedherein by reference in their entirety for all purposes.

Briefly, such alternate constructs include template sequences thatpossess a central double stranded portion that is linked at each end byan appropriate linking oligonucleotide, such as a hairpin loop segment.Such structures not only provide the ability to repeatedly replicate asingle molecule (and thus sequence that molecule), but also provide foradditional redundancy by replicating both the sense and antisenseportions of the double stranded portion. In the context of sequencingapplications, such redundant sequencing provides great advantages interms of sequence accuracy.

The nucleic acids can comprise a population of nucleic acids havinguniversal sequence regions which are common to all of the nucleic acidsin the population and also have specific regions which are different inthe different members of the population. The current invention allowsfor capturing and isolating polymerase-nucleic acid complexes usingeither the universal or the specific regions.

Polymerase enzymes useful in the invention include polymerases mutatedto have desirable properties for sequencing. For example, suitableenzymes include those taught in, e.g., Copending Patent Applicationentitled “Recombinant Polymerases for Incorporation of Protein ShieldNucleotide Analogs”, Attorney Docket No. 105-016900, filed Feb. 14,2013, WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATIONby Hanzel et al., WO 2008/051530 POLYMERASE ENZYMES AND REAGENTS FORENHANCED NUCLEIC ACID SEQUENCING by Rank et al., and U.S. patentapplication Ser. No. 12/584,481 filed Sep. 4, 2009, by Pranav Patel etal. entitled “ENGINEERING POLYMERASES AND REACTION CONDITIONS FORMODIFIED INCORPORATION PROPERTIES.” The modified polymerases may havemodified properties such as decreased branch fraction formation,improved specificity, improved processivity, altered rates, improvedretention time, improved stability of the closed complex, etc.

In addition, the polymerases can also be modified forapplication-specific reasons, such as to increase photostability, e.g.,as taught in U.S. patent application Ser. No. 12/384,110 filed Mar. 30,2009, by Keith Bjornson et al. entitled “Enzymes Resistant toPhotodamage,” to improve activity of the enzyme when bound to a surface,as taught, e.g., in WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES byHanzel et al. and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TOOPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al., or toinclude purification or handling tags as is taught in the citedreferences and as is common in the art. Similarly, the modifiedpolymerases described herein can be employed in combination with otherstrategies to improve polymerase performance, for example, reactionconditions for controlling polymerase rate constants such as taught inU.S. patent application Ser. No. 12/414,191 filed Mar. 30, 2009, andentitled “Two slow-step polymerase enzyme systems and methods,”incorporated herein by reference in its entirety for all purposes.

The polymerase enzymes used in the invention will generally havestrand-displacement activity. Many polymerases have this capability, andit is useful in the context of the current invention for opening up andexposing the regions of a nucleic acid sample for capture by a hookmolecule. In some cases, strand displacement is part of the polymeraseenzyme itself. In other cases, other cofactors or co-enzymes can beadded to provide the strand displacement capability.

The enzymes used in the invention can comprise DNA polymerases. DNApolymerases are sometimes classified into six main groups based uponvarious phylogenetic relationships, e.g., with E. coli Pol I (class A),E. coli Pol II (class B), E. coli Pol III (class C), Euryarchaeotic PolII (class D), human Pol beta (class X), and E. coli UmuC/DinB andeukaryotic RAD30/xeroderma pigmentosum variant (class Y). For a reviewof recent nomenclature, see, e.g., Burgers et al. (2001) “Eukaryotic DNApolymerases: proposal for a revised nomenclature” J Biol. Chem.276(47):43487-90. For a review of polymerases, see, e.g., Hübscher etat. (2002) “Eukaryotic DNA Polymerases” Annual Review of BiochemistryVol. 71: 133-163; Alba (2001) “Protein Family Review: Replicative DNAPolymerases” Genome Biology 2(1): reviews 3002.1-3002.4; and Steitz(1999) “DNA polymerases: structural diversity and common mechanisms” JBiol Chem 274:17395-17398. The basic mechanisms of action for manypolymerases have been determined. The sequences of literally hundreds ofpolymerases are publicly available, and the crystal structures for manyof these have been determined, or can be inferred based upon similarityto solved crystal structures for homologous polymerases. For example,the crystal structure of Φ29, a preferred type of parental enzyme to bemodified according to the invention, is available.

In addition to wild-type polymerases, chimeric polymerases made from amosaic of different sources can be used. For example, Φ29 polymerasesmade by taking sequences from more than one parental polymerase intoaccount can be used as a starting point for mutation to produce thepolymerases of the invention. Chimeras can be produced, e.g., usingconsideration of similarity regions between the polymerases to defineconsensus sequences that are used in the chimera, or using geneshuffling technologies in which multiple Φ29-related polymerases arerandomly or semi-randomly shuffled via available gene shufflingtechniques (e.g., via “family gene shuffling”; see Crameri et al. (1998)“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution” Nature 391:288-291; Clackson et al. (1991) “Makingantibody fragments using phage display libraries” Nature 352:624-628;Gibbs et al. (2001) “Degenerate oligonucleotide gene shuffling (DOGS): amethod for enhancing the frequency of recombination with familyshuffling” Gene 271:13-20; and Hiraga and Arnold (2003) “General methodfor sequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296). In these methods, the recombination points can bepredetermined such that the gene fragments assemble in the correctorder. However, the combinations, e.g., chimeras, can be formed atrandom. For example, using methods described in Clarkson et al., fivegene chimeras, e.g., comprising segments of a Phi29 polymerase, a PZApolymerase, a M2 polymerase, a B103 polymerase, and a GA-1 polymerase,can be generated. Appropriate mutations to improve branching fraction,increase closed complex stability, or alter reaction rate constants canbe introduced into the chimeras.

Available DNA polymerase enzymes have also been modified in any of avariety of ways, e.g., to reduce or eliminate exonuclease activities(many native DNA polymerases have a proof-reading exonuclease functionthat interferes with, e.g., sequencing applications), to simplifyproduction by making protease digested enzyme fragments such as theKlenow fragment recombinant, etc. As noted, polymerases have also beenmodified to confer improvements in specificity, processivity, andimproved retention time of labeled nucleotides inpolymerase-DNA-nucleotide complexes (e.g., WO 2007/076057 POLYMERASESFOR NUCLEOTIDE ANALOGUE INCORPORATION by Hanzel et al. and WO2008/051530 POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEIC ACIDSEQUENCING b_(y) Rank et al.), to alter branch fraction andtranslocation (e.g., U.S. patent application Ser. No. 12/584,481 filedSep. 4, 2009, by Pranav Patel et al. entitled “ENGINEERING POLYMERASESAND REACTION CONDITIONS FOR MODIFIED INCORPORATION PROPERTIES”), toincrease photostability (e.g., U.S. patent application Ser. No.12/384,110 filed Mar. 30, 2009, by Keith Bjornson et al. entitled“Enzymes Resistant to Photodamage”), and to improve surface-immobilizedenzyme activities (e.g., WO 2007/075987 ACTIVE SURFACE COUPLEDPOLYMERASES by Hanzel et al. and WO 2007/076057 PROTEIN ENGINEERINGSTRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzelet al.). Any of these available polymerases can be modified inaccordance with the invention to decrease branching fraction formation,improve stability of the closed polymerase-DNA complex, and/or alterreaction rate constants.

Many such polymerases that are suitable for modification are available,e.g., for use in sequencing, labeling and amplification technologies.For example, human DNA Polymerase Beta is available from R&D systems.DNA polymerase I is available from Epicenter, GE Health Care,Invitrogen, New England Biolabs, Promega, Roche Applied Science, SigmaAldrich and many others. The Klenow fragment of DNA Polymerase I isavailable in both recombinant and protease digested versions, from,e.g., Ambion, Chimerx, eEnzyme LLC, GE Health Care, Invitrogen, NewEngland Biolabs, Promega, Roche Applied Science, Sigma Aldrich and manyothers. Φ29 DNA polymerase is available from e.g., Epicentre. Poly Apolymerase, reverse transcriptase, Sequenase, SP6 DNA polymerase, T4 DNApolymerase, T7 DNA polymerase, and a variety of thermostable DNApolymerases (Taq, hot start, titanium Taq, etc.) are available from avariety of these and other sources. Recent commercial DNA polymerasesinclude Phusion™ High-Fidelity DNA Polymerase, available from NewEngland Biolabs; GoTaq® Flexi DNA Polymerase, available from Promega;RepliPHI™ Φ29 DNA Polymerase, available from Epicentre Biotechnologies;PfuUltra™ Hotstart DNA Polymerase, available from Stratagene; KOD HiFiDNA Polymerase, available from Novagen; and many others.Biocompare(dot)com provides comparisons of many different commerciallyavailable polymerases.

DNA polymerases that are preferred substrates for mutation to decreasebranching fraction, increase closed complex stability, or alter reactionrate constants include Taq polymerases, exonuclease deficient Taqpolymerases, E. coli DNA Polymerase 1, Klenow fragment, reversetranscriptases, Φ29 related polymerases including wild type Φ29polymerase and derivatives of such polymerases such as exonucleasedeficient forms, T7 DNA polymerase, T5 DNA polymerase, an RB69polymerase, etc.

In one aspect, the polymerase that is modified is a Φ29-type DNApolymerase. For example, the modified recombinant DNA polymerase can behomologous to a wild-type or exonuclease deficient Φ29 DNA polymerase,e.g., as described in U.S. Pat. Nos. 5,001,050, 5,198,543, or 5,576,204.Alternately, the modified recombinant DNA polymerase can be homologousto other Φ29-type DNA polymerases, such as B103, GA-1, PZA, 015, BS32,M2Y, Nf, G1, Cp-1, PRD1, PZE, SFS, Cp-5, Cp-7, PR4, PR5, PR722, L17,Φ21, or the like. For nomenclature, see also, Meijer et al. (2001) “Φ29Family of Phages” Microbiology and Molecular Biology Reviews,65(2):261-287. Suitable polymerases are described, for example, in U.S.patent application Ser. Nos. 12/924,701, filed Sep. 30, 2010; and12/384,112, filed Mar. 30, 2009.

In some embodiments, the polymerase enzyme that is used for sequencingis an RNA polymerase including an RNA dependent RNA polymerase and a DNAdependent RNA polymerase. Any suitable RNA polymerase can be usedincluding RNA polymerases from bacteria, eukaryotes, viruses, or archea.Suitable RNA polymerases include RNA PoI I, RNA PoI II, RNA Pol III, RNAPoI IV, RNA PoI V, T7 RNA polymerase, T3 RNA polymerase or SP6 RNApolymerase. Where RNA polymerases are used, the polymerizing reagentswill generally include NTPs or their analogs rather than the dNTPs usedfor DNA synthesis. In addition, RNA polymerases can be used withspecific cofactors. There are many proteins that can bind to RNAP andmodify its behavior. For instance, GreA and GreB from E. coli and inmost other prokaryotes can enhance the ability of RNAP to cleave the RNAtemplate near the growing end of the chain. This cleavage can rescue astalled polymerase molecule, and is likely involved in proofreading theoccasional mistakes made by RNAP. A separate cofactor, Mfd, is involvedin transcription-coupled repair, the process in which RNAP recognizesdamaged bases in the DNA template and recruits enzymes to restore theDNA. Other cofactors are known to play regulatory roles; i.e. they helpRNAP choose whether or not to express certain genes. RNA dependent RNApolymerases (RNA replicases) may also be used including viral RNApolymerases: e.g. polioviral 3Dpol, vesicular stomatitis virus L, andhepatitis C virus NS5b protein; and eukaryotic RNA replicases which areknown to amplify microRNAs and small temporal RNAs and producedouble-stranded RNA using small interfering RNAs as primers.

The use of an RNA dependent polymerase such as an RNA dependent DNApolymerase or an RNA dependent RNA polymerase allows for the directsequencing of messenger RNA, transfer RNA, non-coding RNA, ribosomalRNA, micro RNA or catalytic RNA. The polymerase enzyme used in themethods or compositions of the invention include RNA dependent DNApolymerases or reverse transcriptases. Suitable reverse transcriptaseenzymes include HIV-1, M-MLV, AMV, and Telomere Reverse Transcriptase.Reverse transcriptases also allow for the direct sequencing of RNAsubstrates such as messenger RNA, transfer RNA, non-coding RNA,ribosomal RNA, micro RNA or catalytic RNA.

Thus, any suitable polymerase enzyme can be used in the systems andmethods of the invention. Suitable polymerases include DNA dependent DNApolymerases, DNA dependent RNA polymerases, RNA dependent DNApolymerases (reverse transcriptases), and RNA dependent RNA polymerases.

The conditions required for nucleic acid synthesis are well known in theart. The polymerase reaction conditions include the type andconcentration of buffer, the pH of the reaction, the temperature, thetype and concentration of salts, the presence of particular additiveswhich influence the kinetics of the enzyme, and the type, concentration,and relative amounts of various cofactors, including metal cofactors.

Enzymatic reactions are often run in the presence of a buffer, which isused, in part, to control the pH of the reaction mixture. Bufferssuitable for the invention include, for example, TAPS(3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine(N,N-bis(2-hydroxyethyl)glycine), TRIS (tris(hydroxymethyl)methylamine),ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid), Tricine(N-tris(hydroxymethyl)methylglycine), HEPES4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), and MES(2-(N-morpholino)ethanesulfonic acid).

The pH of the reaction can influence the rate of the polymerasereaction. The temperature of the reaction can be adjusted to enhance theperformance of the system. The reaction temperature may depend upon thetype of polymerase which is employed.

As used in the art, the term nucleotide refers both to the nucleosidetriphosphates that are added to a growing nucleic acid chain in thepolymerase reaction, and also to refer to the individual units of anucleic acid molecule, for example the units of DNA and RNA. Herein, theterm nucleotide used in consistent with its use in the art. Whether theterm nucleotide refers to the substrate molecule to be added to thegrowing nucleic acid or to the units in the nucleic acid chain can bederived from the context in which the term used.

The nucleotides or set of nucleotides used during nucleic acid synthesisare generally naturally occurring nucleotides but can also includemodified nucleotides (nucleotide analogs). The nucleotides used in theinvention, whether natural, unnatural, modified or analog are suitablefor participation in the polymerase reaction. The term nucleotide mayalso be used to refer to nucleotides having other than three phosphategroups, for example 4, 5, 6, 7 or more phosphate groups. Suchnucleotides have been described, for example in U.S. Pat. Nos. 6,936,702and 7,041,812. Labels such as fluorescent dye group may be located invarious positions on the nucleotide. In some cases, a fluorescent dye islocated on the terminal phosphate of the nucleotide.

The nucleotide compositions may include nucleoside triphosphates, oranalogs of such compounds. For example, in some cases, the reactionmixtures will include nucleotide analogs having longer phosphate chains,such as nucleoside tetra, penta-, hexa- or even heptaphosphates. Inaddition, the nucleotide analogs of the compositions of the inventionmay additionally include other components, such as detectable labelinggroups. Such detectable labeling groups will typically impart anoptically or electrochemically detectable property to the nucleotideanalogs being incorporated into the synthesis reaction. In particularlypreferred aspects, fluorescent labeling groups, i.e., labeling groupsthat emit light of one wavelength when excited with light of anotherwavelength, are used as the labeling groups. For purposes of the presentdisclosure, the foregoing or later discussed nucleotide or nucleotideanalog compositions whether labeled or unlabeled, possessing of one ormore phosphate groups, typically two or more or three or more phosphategroups, or otherwise modified, are generally referred to herein asnucleotides.

The methods, compositions, and devices of the invention are particularlyuseful for performing single-molecule analysis. In some cases, thesubstrate or chip comprises an array of nanoscale wells such as arraysof zero mode waveguides (ZMWs). For example, the substrate can have atransparent lower layer such as fused silica, upon which is deposited acladding layer with a thickness of between about 10 nm and about 500 nm.Through the cladding layer is an array of holes extending to thetransparent substrate, and in some cases extending into the transparentsubstrate. The holes can have any suitable profile including a circularprofile. Where the holes have a circular profile, the diameter of theholes is generally from about 20 nm to about 500 nm. The holes extendingto the transparent substrate will generally have a portion of thetransparent substrate as their base, thus forming nanoscale wells. Foruse in the present invention, the arrays of nanoscale wells aretypically functionalized such that binding molecules are attached at thebase of the wells for binding the molecule of interest such as thepolymerase-nucleic acid complex within the well, In some cases, thearrays are selectively functionalized such that a higher density ofbinding molecules is present within the wells than outside of the wells.Approaches to functionalizing zero mode waveguide substrates areprovided in U.S. Pat. Nos. 7,833398, 7,292,742 and in U.S. patentapplication Ser. Nos. 11/731,748, filed March, 29, 2007, 12/079,922,filed Mar. 27, 2008, and 12/074,716, filed Mar. 5, 2008, the fulldisclosures of which are incorporated by reference herein for allpurposes. As described elsewhere herein, these nanoscale wells providefor carrying out analyses on very small numbers of molecules down tosingle molecules. In some cases the methods, devices, and compositionsof the invention allow for the deposition of single molecules ofinterest within nanoscale wells.

The coupling groups or binding molecules on the substrate for couplingbetween the molecule of interest, e.g. polymerase-nucleic acid complexcan be any suitable coupling group or binding molecules. The couplingcan be accomplished by forming a covalent bond or through a non-covalentinteraction. It is generally desired that the coupling to the substrateresult in a strong bond relative to the other linkages. Many types ofbinding pairs are known in the art. In some cases, an interactionbetween biotin and a biotin binding protein such as avidin orstreptavidin is used. In some cases, an antibody-antigen interaction,for example between digoxigenin and anti-digoxigenin is used. Reactionsthat form covalent linkages, for example SNAP or Click chemistry can beused to bind the polymerase-nucleic acid complex to the substrate.Oligonucleotide hybridization can also be used for the attachment. Wheresuch hybridization is used, the linkages are designed such that theoligonucleotide binding to the surface is stronger, e.g. has a higher Tmthan the other linkages between the surface and the bead.

Binding of the polymerase-nucleic acid complex to the substrate can becarried out by forming a bond to the polymerase. One member of thebinding pair used to attach the complex to the solution is connecteddirectly or indirectly to the polymerase. In some cases, a biotinylationsequence is included when producing the polymerase, the protein isbiotinylated and attached to streptavidin prior to formation of thecomplex. The polymerase-streptavidin is then ready for binding to asubstrate that is prepared by having biotin groups on its surface.

Where the molecule of interest comprises a polymerase-nucleic acidcomplex, the solution that is used for deposition with beads isgenerally an aqueous solution. The components of the solution and theconditions are controlled as described above in order that thepolymerase-nucleic acid complex remains intact. For example, theappropriate level of monovalent and divalent ions, the concentration ofnucleotide, the pH and the temperature are controlled. It is alsogenerally desired that the polymerase not continue to perform nucleicacid synthesis during deposition, and Sr and Ca can be added in order toinhibit or reduce polymerization.

One object of the invention is providing molecules of interest such aspolymerase-nucleic acid complexes to a substrate for single moleculeanalysis. For single molecule analysis it is generally desired thatsingle molecules of interest are bound to a substrate at a density andpattern such that the optical signal from one molecule can be detecteddistinctly from signals from other molecules and from solution. That is,the molecules are deposited so as to be individually opticallyresolvable. One method that has been used for this purpose is to depositmolecules of interest from a solution that is diluted such that onaverage, an acceptable number of single molecules will be individuallyoptically resolvable. If the concentration is too high, the density onthe surface will be such that few, if any, single molecules will beresolvable. If the concentration is too low, this may also result invery few single molecules. The methods, devices and compositions of thepresent invention provide an alternative approach for obtaining highlevels of optically resolvable single molecules on a substrate.

As described above, a preferred substrate for single-molecule analysisis a zero mode waveguide (ZMW) array. Here, the optical analysis iscarried out only within the ZMWs on the surface. We have found that theinvention provides useful methods for loading single-molecules into aZMW array. As with other substrates for single molecule analysis,loading molecules of interest onto ZMWs to obtain acceptable numbers ofsingle molecules is often carried out with the dilution method. Themethods of the invention provides tools for controlling the way in whichmolecules of interest are loaded into ZMWs.

When depositing a library of polymerase-nucleic acid complexes onto asubstrate, for example a ZMW substrate, by diffusion from solution wehave found that there can be relatively a large number of smallerfragments deposited than larger fragments. We have found that bydepositing with beads, there is a much more even distribution ofdeposited polymerase-nucleic acid complexes by size, allowing for abetter representation of the larger size fragments in the data in singlemolecule analysis.

Since ZMWs are wells with defined dimensions, the sizes, shapes, andextension (reach) of the beads can be used to control the manner inwhich molecules of interest are deposited. For example in some cases,beads are used that have a size that is smaller than the ZMW, such thatit fits into the ZMW, and has a reach such that only molecules ofinterest from a bead fitting into the ZMW will be deposited. In somecases, beads will be used that are smaller than the diameter of a ZMW,but larger than half of the diameter of the ZMW. In this way, only onebead will deposit into the ZMW, preventing the deposition of a secondbead, ensuring that each ZMW will only receive molecules of interestfrom one bead. For example, where a ZMW array having ZMWs with diametersof 200 nm, beads having diameters from about 100 nm to about 190 nm areused. Another way of controlling the level of loading is by controllingthe density of molecules of interest on the surfaces of the beads. Forexample, by using sparsely functionalized beads, only small numbers ofmolecules of interest will be deposited.

When loading a surface for single molecule analysis, generally a smallamount of material is deposited as compared to the total amount on thebead. This allows for re-using the beads by removing them from thesubstrate, optionally storing them, and then applying them to anothersubstrate. The beads can be re-used in some case to load substrates 1,2, 3, 4, 5, 10, 20 or more times while still obtaining acceptableloading. We have found that after each loading, the amount loaded ontothe next substrate may be slightly less, but that the levels on thelater substrates are still acceptable. Comparable levels can also beobtained on later substrates by changing deposition conditions, forexample by lengthening the time of deposition. The ability to re-use thebeads can be important for getting the most out of small samples. Theability to store the beads for future loading and testing can beimportant for the integrity of the date from a study. We have also foundthat the beads can be stored for days, weeks, and for over a monthwithout any measurable deterioration in properties.

The methods, devices, and compositions of the invention are particularlyuseful for single molecule sequencing, and specifically single moleculesequencing by incorporation in real time. For sequencing processes thatrely upon monitoring of the incorporation of nucleotides into growingnascent strands being synthesized by the complex, the progress of thereaction through these steps is of significant importance. Inparticular, for certain “real-time” nucleotide incorporation monitoringprocesses, the detectability of the incorporation event is improvedbased upon the amount of time the nucleotide is incorporated into andretained within the synthesis complex during its ultimate incorporationinto a primer extension product.

By way of example, in certain exemplary processes, the presence of thenucleotide in the synthesis complex is detected either by virtue of afocused observation of the synthesis complex, or through the use ofinteractive labeling techniques that produce characteristic signals whenthe nucleotide is within the synthesis complex. See, e.g., Levene, etal., Science 299:682-686, January 2003, and Eid, J. et al., Science,323(5910), 133-138 (2009), the full disclosures of which areincorporated herein by reference in their entirety for all purposes;

In the first exemplary technique, as schematically illustrated in FIG.29, a nucleic acid synthesis complex, including a polymerase enzyme2902, a template sequence 2904 and a complementary primer sequence 2906,is provided immobilized within an observation region 2900, that permitsillumination (as shown by hv) and observation of a small volume thatincludes the complex without excessive illumination of the surroundingvolume (as illustrated by dashed line 2908). By illuminating andobserving only the volume immediately surrounding the complex, one canreadily identify fluorescently labeled nucleotides that becomeincorporated during that synthesis, as such nucleotides are retainedwithin that observation volume by the polymerase for longer periods thanthose nucleotides that are simply randomly diffusing into and out ofthat volume.

In particular, as shown in panel II of FIG. 29, when a nucleotide, e.g.,A, is incorporated into by the polymerase, it is retained within theobservation volume for a prolonged period of time, and upon continuedillumination yields a prolonged fluorescent signal (shown by peak 2910).By comparison, randomly diffusing and not incorporated nucleotidesremain within the observation volume for much shorter periods of time,and thus produce only transient signals (such as peak 2912), many ofwhich go undetected, due to their extremely short duration.

In particularly preferred exemplary systems, the confined illuminationvolume is provided through the use of arrays of optically confinedapertures termed zero-mode waveguides, e.g., as shown by confinedreaction region 100 (ZMWs)(See, e.g., U.S. Pat. No. 6,917,726, which isincorporated herein by reference in its entirety for all purposes). Forsequencing applications, the DNA polymerase is provided immobilized uponthe bottom of the ZMW (See, e.g., Korlach et al., PNAS U.S.A. 105(4):1176-1181. (2008), which is incorporated herein by reference in itsentirety for all purposes.

In operation, the fluorescently labeled nucleotides (shown as A, C, Crand T) bear one or more fluorescent dye groups on a terminal phosphatemoiety that is cleaved from the nucleotide upon incorporation. As aresult, synthesized nucleic acids do not bear the build-up offluorescent labels, as the labeled polyphosphate groups diffuses awayfrom the complex following incorporation of the associated nucleotide,nor do such labels interfere with the incorporation event. See, e.g.,Korlach et al., Nucleosides, Nucleotides and Nucleic Acids,27:1072:1083, 2008.

In another exemplary technique, the nucleotides to be incorporated areeach provided with interactive labeling components that are interactivewith other labeling components provided coupled to, or sufficiently nearthe polymerase (which labels are interchangeably referred to herein as“complex borne”). Upon incorporation, the nucleotide borne labelingcomponent is brought into sufficient proximity to the complex-borne (orcomplex proximal) labeling component, such that these components producea characteristic signal event. For example, the polymerase may beprovided with a fluorophore that provides fluorescent resonant energytransfer (FRET) to appropriate acceptor fluorophores. These acceptorfluorophores are provided upon the nucleotide to be incorporated, whereeach type of nucleotide bears a different acceptor fluorophore, e.g.,that provides a different fluorescent signal. Upon incorporation, thedonor and acceptor are brought close enough together to generate energytransfer signal. By providing different acceptor labels on the differenttypes of nucleotides, one obtains a characteristic FRET-basedfluorescent signal for the incorporation of each type of nucleotide, asthe incorporation is occurring.

In a related aspect, a nucleotide analog may include two interactingfluorophores that operate as a donor/quencher pair or FRET pair, whereone member is present on the nucleobase or other retained portion of thenucleotide, while the other member is present on a phosphate group orother portion of the nucleotide that is released upon incorporation,e.g., a terminal phosphate group. Prior to incorporation, the donor andquencher are sufficiently proximal on the same analog as to providecharacteristic signal, e.g., quenched or otherwise indicative of energytransfer. Upon incorporation and cleavage of the terminal phosphategroups, e.g., bearing a donor fluorophore, the quenching or other energytransfer is removed and the resulting characteristic fluorescent signalof the donor is observable.

In preferred aspects, the synthesis complexes in such reaction mixturesare arrayed so as to permit observation of the individual complexes thatare being so modulated. In arraying individual complexes to beindividually optically resolvable, the systems of the invention willposition the complexes on solid supports such that there is sufficientdistance between adjacent individual complexes as to allow opticalsignals from such adjacent complexes to be optically distinguishablefrom each other.

Typically, such complexes will be provided with at least 50 nm and morepreferably at least 100 nm of distance between adjacent complexes, inorder to permit optical signals, and particularly fluorescent signals,to be individually resolvable. Examples of arrays of individuallyresolvable molecules are described in, e.g., U.S. Pat. No. 6,787,308.

In some cases, individual complexes may be provided within separatediscrete regions of a support, for example on a chip. For example, insome cases, individual complexes may be provided within individualoptical confinement structures, such as zero-mode waveguide cores.Examples of such waveguides and processes for immobilizing individualcomplexes therein are described in, e.g., Published International PatentApplication No. WO 2007/123763, the full disclosure of which isincorporated herein by reference in its entirety for all purposes.

The synthesis complexes are typically provided immobilized upon solidsupports, and preferably, upon supporting substrates. The complexes maybe coupled to the solid supports through one or more of the differentgroups that make up the complex. For example, in the case of nucleicacid polymerization complexes, attachment to the solid support may bethrough an attachment with one or more of the polymerase enzyme, theprimer sequence and/or the template sequence in the complex. Further,the attachment may comprise a covalent attachment to the solid supportor it may comprise a non-covalent association. For example, inparticularly preferred aspects, affinity based associations between thesupport and the complex are envisioned. Such affinity associationsinclude, for example, avidin/streptavidin/neutravidin associations withbiotin or biotinylated groups, antibody/antigen associations,GST/glutathione interactions, nucleic acid hybridization interactions,and the like. In particularly preferred aspects, the complex is attachedto the solid support through the provision of an avidin group, e.g.,streptavidin, on the support, which specifically interacts with a biotingroup that is coupled to the polymerase enzyme.

The sequencing processes, e.g., using the substrates described above andthe synthesis compositions of the invention, are generally exploited inthe context of a fluorescence microscope system that is capable ofilluminating the various complexes on the substrate, and obtainingdetecting and separately recording fluorescent signals from thesecomplexes. Such systems typically employ one or more illuminationsources that provide excitation light of appropriate wavelength(s) forthe labels being used. An optical train directs the excitation light atthe reaction region(s) and collects emitted fluorescent signals anddirects them to an appropriate detector or detectors. Additionalcomponents of the optical train can provide for separation of spectrallydifferent signals, e.g., from different fluorescent labels, anddirection of these separated signals to different portions of a singledetector or to different detectors. Other components may provide forspatial filtering of optical signals, focusing and direction of theexcitation and or emission light to and from the substrate.

One such exemplary system is shown in FIG. 30. An exemplary system isalso described in Lundquist et al., Published U.S. Patent ApplicationNo. 2007-0036511, Optics Letters, Vol. 33, Issue 9, pp. 1026-1028, thefull disclosure of which is incorporated herein by reference in itsentirety for all purposes.

Fluorescence reflective optical trains can be used in the applicationsof the systems of the invention. For a discussion on the advantages ofsuch systems, see, e.g., U.S. patent application Ser. Nos. 11/704,689,filed Feb. 9, 2007, 11/483,413, filed Jul. 7, 2006, and 11/704,733,filed Feb. 9, 2007, the full disclosures of which are incorporatedherein by reference in their entirety for all purpose.

For purposes of the present invention, the processes and systems will bedescribed with reference to detection of incorporation events in a realtime, sequence by incorporation process, e.g., as described in U.S. Pat.Nos. 7,056,661, 7,052,847, 7,033,764 and 7,056,676 (the full disclosuresof which are incorporated herein by reference in their entirety for allpurposes), when carried out in arrays of discrete reaction regions orlocations. An exemplary sequencing system for use in conjunction withthe invention is shown in FIG. 30. As shown, the system includes asubstrate 3002 that includes a plurality of discrete sources of opticalsignals, e.g., reaction wells, apertures, or optical confinements orreaction locations 3004. In typical systems, reaction locations 3004 areregularly spaced and thus substrate 3002 can also be understood as anarray 3002 of reaction locations 3004. The array 3002 can comprise atransparent substrate having cladding layer on its top surface with anarray of nanoscale apertures extending through the cladding to thetransparent substrate. This configuration allows for one or more samplesto be added to the top surface of the array, and for the array to beobserved through the transparent substrate from below, such that onlythe light from the apertures is observed. The array can be illuminatedfrom below as shown in FIG. 30, and in some embodiments, the array canalso be illuminated from above (not shown in FIG. 30).

For illumination from below, one or more excitation light sources, e.g.,lasers 3010 and 3020, are provided in the system and positioned todirect excitation radiation at the various signal sources. Here, twolasers are used in order to provide different excitation wavelengths,for example with one laser 3010 providing illumination in the red, andlaser 3020 providing illumination in the green. The use of multiplelaser excitation sources allows for the optimal excitation of multiplelabels in a sample in contact with the array. The excitationillumination can be a flood illumination, or can be directed to discreteregions on the array, for example, by breaking the excitation beam intoan array of beamlets, each beamlet directed to a feature on the array.In order to break the excitations beams into an array of beamlets, adiffractive optical element (DOE). In the system of FIG. 30, the lightfrom excitation sources 3010 and 3020 is sent through DOE components3012 and 3022 respectively. The use of a DOE for providing an array ofbeamlets is provided, e.g. in U.S. Pat. No. 7,714,303, which isincorporated by reference herein in its entirety. Excitation light isthen passed through illumination relay lenses 3014 and 3024 to interactwith dichroic 3026. In the system of FIG. 30, the red light from laser3010 is reflected off of dichroic 3026, and the green light from laser3020 is directed through the dichroic 3026. The excitation light is thenpassed through illumination tube lens 3028 into objective lens 3070 andonto the array 3002.

Emitted signals from sources 3004 are then collected by the opticalcomponents, e.g., objective 3070, comprising dichroic element 3075 whichallows the illumination light to pass through and reflects theexcitation light. The emitted light passes through collection tube lens3030 and collection relay lens 3032. The emitted light is then separatedinto D different spectral channels, and each spectral channel isdirected to a different detector. In the system of FIG. 30, the light isseparated into four different channels, each channel correspondingpredominantly to one of four labels to be detected in the sample. Thus,the system allows the user to obtain four two dimensional images, eachimage corresponding to one of the four labels. In order to separate thelight into the four spectral channels, dichroics 3040, 3042, and 3044are used. Dichroic 3040 allows the light for channels 1 and 2 to passwhile reflecting the light for channels 3 and 4. Dichroic 3042 allowsthe light for channel 1 to pass, through collection imaging lens 3051 todetector 3061, and reflects the light for channel 2 through collectionimaging lens 3052 to detector 3062. Dichroic 3044 allows the light forchannel 3 to pass, through collection imaging lens 3053 onto detector3063, and reflects the light for channel 4 through collectionillumination lens 3054 onto detector 3064. Each of the detectors3061-3064 comprise arrays of pixels. The detectors can be, for example,CMOS, EMCCD, or CCD arrays. Each of the detectors obtains 2-dimensionalimages of the channel that is directed to that detector. The data fromthose signals is transmitted to an appropriate data processing unit,e.g., computer 3070, where the data is subjected to processing,interpretation, and analysis. The data processing unit is configured toprocess the data both pixel by pixel and pixel region by pixel region,where each pixel region corresponds to a feature on the substrate. Thedata processing unit can receive data from calibration runs in order todefine software mask pixel weighting, spectral weighting, and noiseparameters. These parameters and weightings can be applied to signalsthat are measured on the detectors during an analytical reaction such asduring sequencing. In some embodiments, the data processing unit isconfigured to define and apply software mask pixel weighting, spectralweighting, and noise parameters that are determined and then appliedduring an analytical reaction such as during sequencing.

Analyzed and processed obtained from the analytical reactions canultimately be presented in a user ready format, e.g., on display 3075,printout 3085 from printer 3080, or the like, or may be stored in anappropriate database, transmitted to another computer system, orrecorded onto tangible media for further analysis and/or later review.Connection of the detector to the computer may take on a variety ofdifferent forms. For example, in preferred aspects, the detector iscoupled to appropriate Analog to Digital (A/D) converter that is thencoupled to an appropriate connector in the computer. Such connectionsmay be standard USB connections, Firewire® connections, Ethernetconnections or other high speed data connections. In other cases, thedetector or camera may be formatted to provide output in a digitalformat and be readily connected to the computer without any intermediatecomponents.

This system, and other hardware descriptions herein, are provided solelyas a specific example of sample handling and image capture hardware toprovide a better understanding of the invention. It should beunderstood, however, that the present invention is directed to dataanalysis and interpretation of a wide variety of real-time florescentdetecting systems, including systems that use substantially differentillumination optics, systems that include different detector elements(e.g., EB-CMOS detectors, CCD's, etc.), and/or systems that localize atemplate sequence other than using the zero mode wave-guides describedherein.

In the context of the nucleic acid sequencing methods described herein,it will be appreciated that the signal sources each represent sequencingreactions, and particularly, polymerase mediated, template dependentprimer extension reactions, where in preferred aspects, each baseincorporation event results in a prolonged illumination (orlocalization) of one of four differentially labeled nucleotides beingincorporated, so as to yield a recognizable pulse that carries adistinguishable spectral profile or color.

The present invention can include computer implemented processes, and/orsoftware incorporated onto a computer readable medium instructing suchprocesses, as set forth in greater detail below. As such, signal datagenerated by the reactions and optical systems described above, is inputor otherwise received into a computer or other data processor, andsubjected to one or more of the various process steps or components setforth below. Once these processes are carried out, the resulting outputof the computer implemented processes may be produced in a tangible orobservable format, e.g., printed in a user readable report, displayedupon a computer display, or it may be stored in one or more databasesfor later evaluation, processing, reporting or the like, or it may beretained by the computer or transmitted to a different computer for usein configuring subsequent reactions or data processes.

Computers for use in carrying out the processes of the invention canrange from personal computers such as PC or Macintosh® type computersrunning Intel Pentium or DuoCore processors, to workstations, laboratoryequipment, or high speed servers, running UNIX, LINUX, Windows®, orother systems. Logic processing of the invention may be performedentirely by general purposes logic processors (such as CPU's) executingsoftware and/or firmware logic instructions; or entirely by specialpurposes logic processing circuits (such as ASICs) incorporated intolaboratory or diagnostic systems or camera systems which may alsoinclude software or firmware elements; or by a combination of generalpurpose and special purpose logic circuits. Data formats for the signaldata may comprise any convenient format, including digital image baseddata formats, such as MEG, GIF, BMP, TIFF, or other convenient formats,while video based formats, such as avi, mpeg, mov, rmv, or other videoformats may be employed. The software processes of the invention maygenerally be programmed in a variety of programming languages including,e.g., Matlab, C, C++, C#, NET, Visual Basic, Python, JAVA, CGI, and thelike.

While described in terms of a particular sequencing by incorporationprocess or system, it will be appreciated that certain aspects of theprocesses of the invention may be applied to a broader range ofanalytical reactions or other operations and varying systemconfigurations than those described for exemplary purposes.

In some cases, the compositions, methods, and systems of the inventioncan be used as part of an integrated sequencing system, for example, asdescribed in US 20120014837—Illumination of Integrated AnalyticalSystems, US 20120021525—Optics Collection and Detection System andMethod, US 20120019828—Integrated Analytical System and Method,61/660,776 filed Jun. 17, 2012—Arrays of Integrated Analytical Devicesand Methods for Production, and US 20120085894—Substrates and OpticalSystems and Methods of Use Thereof which are incorporated herein byreference in their entirety for all purposes.

In certain embodiments, the sequencing compositions described hereinwill be provided in whole, or in part, in kit form enabling one to carryout the processes described herein. Such kits will typically compriseone or more components of the reaction complex, such as the polymeraseenzyme and primer sequences. Such kits will also typically includebuffers and reagents that provide the catalytic and non-catalytic metalco-factors employed in the processes described herein. The kits willalso optionally include other components for carrying out sequencingapplications in accordance with those methods described herein. Inparticular, such kits may include ZMW array substrates for use inobserving individual reaction complexes as described herein.

In addition to the various components set forth above, the kits willtypically include instructions for combining the various components inthe amounts and/or ratios set forth herein, to carry out the desiredprocesses, as also described or referenced herein, e.g., for performingsequence by incorporation reactions.

Proteins Having Buried Chromophores

Another approach of the invention for ensuring that the dye portion of anucleotide analog does not come into contact with the polymerase enzymeis to use protein scaffolds that have buried chromophores. In thisapproach, one or more nucleotides is attached to a protein scaffold thathas one or more chromophores within its protein structure. By the termburied or the term within the protein structure, we mean that the dyesare surrounded by amino acid residues in the protein so that they arenot accessible for contact with a molecule that could come into contactwith the protein. Natural proteins are known that have chromophoresburied within the protein. One such protein is allophycocyanin, which isthe primary pigment-protein component of the cores of the phycobilisomeantenna complex. See McGregor, et al. Journal: (2008) J. Mol. Biol. 384:406-421, incorporated herein by reference in its entirety. A threedimensional representation of allophycocyanin is shown in FIG. 12.Allophycocyanin has three chromophores within its structure. One or moreresidues can be mutated to provide attachment points for one or morenucleotide residues.

Another protein scaffold having buried chromophores is green fluorescentprotein (GFP). GFP is a protein composed of about 238 amino acidresidues (26.91(1)a) that exhibits bright green fluorescence whenexposed to ultraviolet blue light. In addition to native GFP, otherrelated fluorescent proteins can also be used. For example, many mutantsof GFP have been produced which have fluorescence at differentwavelengths to GFP. See e.g. Shaner, N. et al. Nat Methods 2 (12):905-9, incorporated herein by reference in its entirety. Amino acids onthe protein or mutated into the protein can be used for attachment ofone or more nucleotide analogs, resulting in a nucleotide analog havinga fluorescent dye that will not come into contact with a polymeraseassociated with the nucleotide portion of the analog. For sequencingapplications, several different GFP type proteins can be used to providespectral separation in order to distinguish the bases for providing thesequence of the template. Suitable GFP type proteins include mPlum,mCherry, tdTomato, mStrawberry, DsRed-monomer, mOrange, mKO, mCitrine,Venus, YPet, EYFP, Emerald, EGFP, CyPet, mCFPm, Cerulean, andT-Sapphire. FIG. 13 shows a three dimensional representation of a GFPshowing the buried natural chromophore. Lysines and cysteines, shown asspheres, represent positions to which one or more nucleotides can beattached as described herein.

Other proteins that can be used include phycobiliproteins, ferritin,phycoerythrin or phycohemerythrin. These can be used with their nativechromophore or the protein can be modified to include non-nativechromophores having desired properties.

Multi-Level Dye Analogs to Mitigate Pulse Merging

One aspect of the invention is the use of a set of analogs wherein eachof the types of nucleotide analogs is represented by multiple analogs,each having a different intensity level. For example, where there arefour analog types, each representing one of A, G, C, T or A, G, C, U,for each of the types there are analogs present having 1, 2, 3, 4, 5, or6 fluorescent dyes. An advantage of this approach is that it provides away of mitigating pulse merging in real time sequencing. Pulse mergingresults when two real time sequencing events happen close together intime such that it is difficult to tell that they are separate events. Insome cases when the individual pulses are not identified, they are seenas a single “merged” pulse. The use of a mixture of multilevel dyesallows the base caller to use brightness as a method to discriminate onevs. multiple incorporation events in a run of homopolymers. FIG. 31illustrates this concept. The pulse train shown in FIG. 31(A)illustrates a run of three Gs having a short inter pulse distance (IPD)between them. When the pulses merge in this manner, the base caller canhave trouble correctly recognizing that this represents 3 Gs. Theexample shown in FIG. 31(B) illustrates a pulse train with similaroverall kinetics to that in FIG. 31(A), but with the use of multi-leveldyes. Here, rather than having one nucleotide analog representing G, agroup of nucleotide analogs is used in which the members of the grouphave different brightness levels. This can be accomplished, for exampleby having three G analogs, each with a different brightness level. Insome cases, 3, 4, 5, 6, 7, 9, or 10 different brightness levels can beused. The group of analogs can be a set of analogs each using the sametype of dye, but with different members of the set having differentnumbers of dyes.

Multilevel dye analogs can be made, for example using a streptavidincore. For example one or more nucleotides can be connected tostreptavidin through one of the four biotin binding sites. The otherthree sites can be randomly populated with dye such that a set ofnucleotide analogs (each having one type of base, e.g. G) having 1, 2and 3 dyes or 2, 4, and 6 dyes is created. Alternatively, the one ormore nucleotide moieties can be attached directly to the streptavidin,and the four biotin binding sites can be randomly populated with dyes toprovide an analog set having 1, 2, 3, and 4 dyes, or 2, 4, 6, and 8dyes. Multilevel dye nucleotide analogs with a streptavidin core havebeen produced by the inventors and used in real time single moleculesequencing. Results showed that analogs having multiple different levelsof signal could be detected.

EXAMPLES Example 1 Sequencing with Nucleotide Analogs Using aStreptavidin Protein Shield

FIG. 32 shows a type of streptavidin structures that can be used toincrease photostability of a nucleic acid sequencing reaction. Thecentral portion of the analog is the tetrameric streptavidin protein. Adye is attached through a linker molecule having two biotin sites thatare designed such that each of the biotins can attach to one bindingsite on a single streptavidin. To the other two sites on thestreptavidin are attached nucleotides (nucleoside phosphates, Pn-NS)that are attached to the streptavidin through their phosphate groups(Pn). In the version shown in FIG. 32, two nucleotides are attached ateach of the two biotin binding sites resulting in four nucleosidephosphates per streptavidin. Analogous constructs can be made having 2,3, 4, or more dyes attached to a the linker molecule having two biotins.The number of nucleotides can also be varied by having 1, 2, 3, 4 ormore nucleotides bound to each biotin binding site.

FIG. 33 shows one example of a structure of a nucleotide analog having aprotein shield that the inventors have prepared and used to performsingle molecule real time nucleic acid sequencing. TheA555-T1-bis-biotin shown in FIG. 33 was synthesized by conventionalorganic chemical techniques. A555 is a fluorescent dye moiety that canbe useful in single molecule sequencing. T1 is the designation of thetrifunctional unit. The A555-T1-bis-biotin was reacted with streptavidinand purified to isolate the complex having a single A555-T1-bis-biotinattached to streptavidin. This complex was then contacted withbiotin-bis-dT6P (shown in FIG. 33). Biotin-bis-dT6P has a single biotinattached through a trifunctional liner to two nucleotides. Thenucleotides are deoxythymidine hexaphosphates. This synthesis results inthe preparation of the nucleotide analog shown in FIG. 33 having oneA555 dye on one side of the streptavidin and having four nucleotidesattached to the other side of the streptavidin.

The nucleotide analog with protein shield of FIG. 33 was used in asingle molecule sequencing reaction carried out in a sequencing systemas describe in Eid, J. et al., Science, 323(5910), 133-138 (2009)) usinga 72 base circular template. The experiment was carried out to emphasizephotodamage in the channel with the protein shield nucleotide analog.The movie length was 30 minutes, and the laser power was 2.5 μW/μm².Concentrations were adjusted, e.g. by raising the concentrations of theprotein shield nucleotide analog to match the kinetics (e.g. interpulsedistance) of a control reaction carried out in the same manner, but witha conventional nucleotide analog in place of the protein shieldnucleotide analog. Results of the experiment are shown in FIGS. 19 and20. A measure of improvements in photostability of the sequencing systemis the readlength. Experiments have shown that when light exposure isminimized, longer readlengths can be obtained, indicating thatphotodamage is limiting the readlength. FIG. 34 shows readlengthhistograms for the control (A) and protein shielded nucleotide analog(B). It can be seen from the figures that the reaction run with theprotein shielded nucleotide analog has a distribution with significantlylonger readlengths. The change in shape of the histograms is partly dueto the fact that for longer read lengths, the length of the movie can bethe limiting factor in the readlength obtained. FIG. 35 shows the datafor four control movies (A) and four protein shielded nucleotide analogmovies (B). Tau (τ) is a measure of the readlength. It can be seen thatfor the control, τ is 1,348 base pairs, while τ for the protein shieldnucleotide analog is 4,037 base pairs. Again, this shows a significantincrease in readlength which indicates a significant improvement inphotostability of the sequencing system by using the protein shieldednucleotide analog.

Example 2 Streptavidin Constructs

The following streptavidin constructs have been cloned for use asprotein shields:

pET11a.Core_Streptavidin.co pET11a.CTerm_His6co.Core_Streptavidin.copET11a.Core_Streptavidin.N23A_S27D_S45A.copET11a.Core_Streptavidin.N23A_S27D_S45A_N49C.copET11a.Core_Streptavidin.N23A_S27D_S45A_S139C.copET11a.Core_Streptavidin.N49C.co pET11a.Core_Streptavidin.S139C.co

Example 3 Top7 Constructs

The following mutants have been cloned:

pET11a.TOP7.co.His6copET11a.TOP7.K15R_K31R_K41R_K42R_K46R_K57R_K58R_K62R_K69R_110.1C.co.His6copET11a.BtagV07co.TOP7.100.1C.co.His6co

The first three mutants have been purified, and found to express well.The second construct knocks out all the lysines except for theN-terminus, and introduces a unique cysteine. This second constructgenerates a scaffold which can be attached to one dye and one base, withthe two attachment points ˜30 Angstroms apart. The third constructallows multiple attachment points through lysine (e.g. for bases) and aunique cysteine through which to couple a dye. Alternately, because itcontains a B-tag, this construct can also be combined with thestreptavidin experiments. For example using biotin tagged dye+lysinetargeted base tagged top 7, one could obtain streptavidin tetramerswhere some of the monomers were associated with dye, and other monomerswith top7/base.

Example 4 Real-Time Single Molecule Sequencing Using Nucleotide Analogswith Both the Dye Component and Phospholinked Nucleotide ComponentAttached Through a Bis-Biotin Moiety

This experiment was performed to determine the sequencing performance ofa full set of four protein shield nucleotide analogs with both the dyecomponent and phospholinked nucleotide component attached throughbis-biotin linkers. A set of protein shield analogs was made with astructure similar to that shown in FIG. 23 corresponding to bases A, G,C, and T. Each of the T, A, and C protein shield nucleotide analogs had6 phospholinked nucleotide moieties linked to a streptavidin through abis-biotin linker. The G protein shield nucleotide analogs had 4phospholinked nucleotide moieties linked to a streptavidin through abis-biotin linker. Each of the nucleotide analogs had a different dyecomponent. The T analog had a double dye moiety with an emission maximumof about 558 nm, the G analog had a FRET dye pair with an emissionmaximum of about 598 nm, the A analog had a single dye with an emissionmaximum of about 659 nm, and the C analog had a FRET dye pair with anemission maximum of about 697 nm. The four protein shield nucleotideanalogs were added at a final concentration of 65-1,000 nM. The templatenucleic acid was a 250 base pair cyclic template. Sequencing wasperformed on a PACBIO™ RS sequencing instrument using standard laser andanalysis options. The SMRT sequencing method performed is described, forexample, in Korlach et al., Methods in Enzymology, Volume 472, 2010,Pages 431-455. In addition, free streptavidin was added to the mix inthe range of 65 to 650 nM. Additionally 650 nM free streptavidin isadded to the chip wash buffer. All other reagents are standard. Thepolymerase enzyme used was a mutant Phi-29 polymerase enzyme includingan E508R mutation such as described in copending patent applicationentitled “Recombinant Polymerases for Incorporation of Protein ShieldNucleotide Analogs”, Attorney Docket No. 105-016900, filed Feb. 14,2013, which is incorporated herein by references in its entirety for allpurposes. The sequencing movies were run for two hours. The proteinshield nucleotide analogs performed very well with an average readlengthwas 9,650 bases with an average accuracy of 83.8%. The fraction oftraces that were movie limited was 25%.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually and separately indicated to beincorporated by reference for all purposes.

We claim:
 1. A labeled nucleotide analog comprising: an avidin proteinhaving four subunits, each subunit comprising one biotin binding site;one or two nucleotide components bound to the avidin protein, eachcomprising one or more phospholinked nucleotide moieties; and one or twodye components, bound to the avidin protein, each comprising one or moredye moieties; wherein each component is bound to the avidin proteinthrough a biotin moiety attached to a binding site on the avidinprotein; and wherein at least one of the nucleotide or dye componentscomprise a bis-biotin moiety bound to two of the biotin binding sites onthe avidin protein.
 2. The labeled nucleotide analog of claim 1 whereinthe nucleotide analog has one nucleotide component that comprises abis-biotin moiety, and the labeled nucleotide analog has two dyecomponents each bound to the avidin through a single biotin moiety. 3.The labeled nucleotide analog of claim 1 wherein the nucleotide analoghas one dye component that comprises a bis-biotin moiety, and thelabeled nucleotide analog has two nucleotide components each bound tothe avidin through a single biotin moiety.
 4. The labeled nucleotideanalog of claim 1 wherein the labeled nucleotide analog has one dyecomponent and one nucleotide component and each of the dye component andthe nucleotide component comprises a bis-biotin moiety.
 5. The labelednucleotide analog of claim 1 wherein the number of dye moieties isbetween 1 and 18 and the number of nucleotide moieties is between 1 and18.
 6. The labeled nucleotide analog of claim 1 wherein the number ofdye moieties is 1, 2, or 3 and the number of nucleotide moieties is 6,7, or
 8. 7. The labeled nucleotide analog of claim 1 wherein the numberof bonds between biotins on the bis-biotin moiety is between 15 and 50.8. The labeled nucleotide analog of claim 1 wherein the avidin proteincomprises either streptavidin or tamavidin.
 9. The labeled nucleotideanalog of claim 1 wherein the dye moieties comprise fluorescent labels.10. A reaction mixture for sequencing a nucleic acid templatecomprising: a polymerase enzyme complex comprising a polymerase enzyme,a template nucleic acid, and optionally a primer hybridized to thetemplate nucleic acid, wherein the polymerase enzyme complex isimmobilized on a surface; and sequencing reagents in contact with thesurface comprising reagents for carrying out nucleic acid synthesisincluding 2 or more labeled nucleotide analogs of claim
 1. 11. Thereaction mixture of claim 10 wherein a labeled nucleotide analog has onedye component and one nucleotide component and each of the dye componentand the nucleotide component comprises a bis-biotin moiety.
 12. A methodfor sequencing a nucleic acid template comprising: providing apolymerase enzyme complex comprising a polymerase enzyme, a templatenucleic acid, and optionally a primer hybridized to the template nucleicacid, wherein the polymerase enzyme complex is immobilized on a surface;adding sequencing reagents in contact with the surface comprisingreagents for carrying out nucleic acid synthesis including 2 or morelabeled nucleotide analogs of claim 1; and determining the sequentialaddition of nucleotides to a nucleic acid strand complementary to astrand of the template nucleic acid by observing the interaction of thelabeled nucleotide analogs with the polymerase enzyme complex.
 13. Themethod of claim 12 wherein the nucleotide component comprises abis-biotin moiety, and a labeled nucleotide analog has two dyecomponents each bound to the avidin through a single biotin moiety. 14.The method of claim 12 wherein the dye component comprises a bis-biotinmoiety, and a labeled nucleotide analog has two nucleotide componentseach bound to the avidin through a single biotin moiety.
 15. The methodof claim 12 wherein a labeled nucleotide analog has one dye componentand one nucleotide component and each of the dye component and thenucleotide component comprises a bis-biotin moiety.
 16. The method ofclaim 12 wherein the number of dye moieties is between 1 and 18 and thenumber of nucleotide moieties is between 1 and 18 in each nucleotideanalog.
 17. The method of claim 12 wherein the number of dye moieties is1, 2, or 3 and the number of nucleotide moieties is 6, 7, or 8 in eachnucleotide analog.
 18. The method of claim 12 wherein the number ofbonds between biotins on a bis-biotin moiety is between 15 and
 50. 19.The method of claim 12 wherein the avidin protein in an analog compriseseither streptavidin or tamavidin.
 20. The method of claim 12 wherein thedye moieties comprise fluorescent labels.
 21. A system for sequencingnucleic acids comprising: a chip comprising a plurality of polymeraseenzyme complexes bound thereto, each polymerase enzyme complexindividually optically resolvable, each polymerase enzyme complexcomprising a polymerase enzyme, a template nucleic acid, and optionallya primer hybridized to the template nucleic acid, sequencing reagents incontact with the surface comprising reagents for carrying out nucleicacid synthesis including 2 or more labeled nucleotide analogs of claim1; and an illumination system for illuminating the polymerase enzymecomplexes; and an optical detection system for detecting fluorescencefrom the labeled nucleotide analogs while they are interacting with thepolymerase enzyme complexes; and a computer for analyzing the signalsdetected by the detection system to determine the sequential addition ofnucleotides to a nucleic acid strand complementary to a strand of thetemplate nucleic acid.